[go: up one dir, main page]

WO2025217454A2 - Ionizable cationic lipids and lipid nanoparticles - Google Patents

Ionizable cationic lipids and lipid nanoparticles

Info

Publication number
WO2025217454A2
WO2025217454A2 PCT/US2025/024156 US2025024156W WO2025217454A2 WO 2025217454 A2 WO2025217454 A2 WO 2025217454A2 US 2025024156 W US2025024156 W US 2025024156W WO 2025217454 A2 WO2025217454 A2 WO 2025217454A2
Authority
WO
WIPO (PCT)
Prior art keywords
lipid
cicl
ionizable cationic
cationic lipid
tlnp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/024156
Other languages
French (fr)
Other versions
WO2025217454A3 (en
Inventor
Priya Prakash Karmali
Steven Tanis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Capstan Therapeutics Inc
Original Assignee
Capstan Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Capstan Therapeutics Inc filed Critical Capstan Therapeutics Inc
Publication of WO2025217454A2 publication Critical patent/WO2025217454A2/en
Publication of WO2025217454A3 publication Critical patent/WO2025217454A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/16—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C219/00—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton
    • C07C219/02—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C219/04—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C219/06—Compounds containing amino and esterified hydroxy groups bound to the same carbon skeleton having esterified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having the hydroxy groups esterified by carboxylic acids having the esterifying carboxyl groups bound to hydrogen atoms or to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
    • C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
    • C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C237/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups
    • C07C237/02—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/08—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/04—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/12—Oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/42—Oxygen atoms attached in position 3 or 5
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D211/40—Oxygen atoms
    • C07D211/44—Oxygen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/02—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D223/04—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings with only hydrogen atoms, halogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/02—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D223/06—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D223/08—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • Lipid nanoparticles comprising ionizable cationic lipids have been developed to address these issues to the extent that RNA-based products, such as the siRNA ONPATTRO ® and two mRNA-based SARS-CoV-2 vaccines have received regulatory approval and entered the marketplace.
  • LNP administered intravenously are taken up primarily in the liver, lung, or spleen depending to a significant degree on net charge and particle size. It is possible to direct >90% of LNP to the liver by a combination of formulation and intravenous administration, for example. Intramuscular administration can provide a clinically useful level of local delivery and expression.
  • LNP can be redirected to other tissues or cell types by conjugating to the LNP a binding moiety with specificity for the target tissue or cell type, for example, conjugating an antibody to an LNP (see, e.g., Endsley and Ho, J. Acquir. Immune Defic.
  • this disclosure fulfills the needs for addressing issues of off-target delivery, poor efficiency of release of therapeutic agents and provides further related advantages.
  • this disclosure provides ionizable cationic lipids having a structure of formula M5 set forth herein.
  • this disclosure provides ionizable cationic lipids having a structure of formula M4 set forth herein. [0009] In some embodiments, this disclosure provides ionizable cationic lipids CICL-250, CICL-291, CICL-292, CICL-293, CICL-294, CICL-295, CICL-296, CICL-297, CICL-298, CICL-299, CILC-300, CICL-301, CICL-302, CICL-303, CICL-309, CICL-310, and CICL-311 set forth herein.
  • this disclosure provides ionizable cationic lipids CICL- 250-61, CICL-250-62, CICL-250-63, CICL-250-64, CICL-250-65, CICL-250-66, CICL-250- 67, CICL-250-68, CICL-250-69, CICL-250-70, CICL-250-71, CICL-250-72, CICL-250-73, CICL-250-74, CICL-250-75, CICL-250-76, CICL-250-77, CICL-291-61, CICL-291-62, CICl- 291-63, CICL-291-65, CICL-291-67, CICL-291-68, CICL-291-72, CICL-291-75, CICL-292- 61, CICL-292-62, CICL-292-63, CICL-292-65, CICL
  • this disclosure provides methods of synthesizing ionizable cationic lipids as described herein, e.g. CICL-250, CICL-291, CICL-292, CICL-293, CICL- 294, CICL-295, CICL-296, CICL-297, CICL-298, CICL-299, CILC-300, CICL-301, CICL-302, CICL-303, CICL-309, CICL-310, and CICL-311 set forth herein.
  • CICL-250 e.g. CICL-250, CICL-291, CICL-292, CICL-293, CICL- 294, CICL-295, CICL-296, CICL-297, CICL-298, CICL-299, CILC-300, CICL-301, CICL-302, CICL-303, CICL-309, CICL-310, and CICL-311 set forth herein
  • this disclosure provides methods of synthesizing ionizable cationic lipids as described herein, e.g., CICL-250-61, CICL-250-62, CICL-250-63, CICL- 250-64, CICL-250-65, CICL-250-66, CICL-250-67, CICL-250-68, CICL-250-69, CICL-250- 70, CICL-250-71, CICL-250-72, CICL-250-73, CICL-250-74, CICL-250-75, CICL-250-76, CICL-250-77, CICL-291-61, CICL-291-62, CICl-291-63, CICL-291-65, CICL-291-67, CICL- 291-68, CICL-291-72, CICL-291-75, CICL-292-61, CICL-292-62, CICL-292
  • this disclosure provides intermediates and methods of synthesizing such intermediates useful in the synthesis of the ionizable cationic lipids as described herein.
  • this disclosure provides lipid nanoparticles (LNPs) and targeted lipid nanoparticles (tLNPs) incorporating the ionizable cationic lipids disclosed herein.
  • this disclosure provides methods for preparing LNPs and tLNPs as described herein.
  • this disclosure provides methods of delivering a biologically active payload (e.g., nucleic acid molecules encoding a therapeutic agent) into a cell comprising contacting the cell with an LNP or tLNP of this disclosure.
  • a biologically active payload e.g., nucleic acid molecules encoding a therapeutic agent
  • FIGS. 1A-1C depict the transfection rate (percentage of cells expressing the mRNA) versus expression level (as molecules of equivalent soluble fluorochrome (MESF)) for two tLNP compositions with CICL-250 or CICL-1, as indicated.
  • the full lipid compositions are described in Table 5.
  • LNPs lipid nanoparticles
  • a biologically active payload e.g., nucleic acid molecules encoding a therapeutic agent
  • LNP compositions are also disclosed herein, including LNPs comprising a functionalized PEG-lipid to enable conjugation of a binding moiety to generate targeted LNPs (tLNPs); that is, LNPs containing a binding moiety that directs the tLNP to a desired tissue or cell type (e.g., immune cells such as T cells or stem cells such as hematopoietic stem cells (HSCs)).
  • tLNPs targeted LNPs
  • desired tissue or cell type e.g., immune cells such as T cells or stem cells such as hematopoietic stem cells (HSCs)
  • Also disclosed herein are methods of delivering a nucleic acid molecule into a cell comprising contacting the cell with an LNP or tLNP of this disclosure.
  • the LNP and tLNP of this disclosure can be used for in vivo, ex vivo, or extracorporeal transfection.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range of this disclosure relating to any physical feature, such as polymer subunits, size, or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • “Derivative,” as used herein, refers to a chemically or biologically modified version of a compound that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound.
  • a “derivative” differs from an “analogue” in that a parent compound can be the starting material to generate a “derivative,” whereas the parent compound can not necessarily used as the starting material to generate an “analogue.”
  • a derivative can have different chemical or physical properties than the parent compound. For example, a derivative can be more hydrophilic or hydrophobic, or it can have altered reactivity as compared to the parent compound.
  • a derivative can be obtained by physical (for example, biological or chemical) modification of the parent compound
  • a derivative can also be conceptually derived, for example, as when a protein sequence is designed based on one or more known sequences, an encoding nucleic acid is constructed, and the derived protein obtained by expression of the encoding nucleic acid.
  • extracorporeal is used with reference to cells, such as peripheral blood or bone marrow cells, harvested or extracted from the body and the manipulation or modification of those cells prior to their intended return (reinfusion).
  • Manipulation and modification of cells generally relates to cell separation and washing procedures and exposure to activation agents (e.g., biological response modifiers (BRMs)) and transfection agents (e.g., LNPs, tLNPs), over a time interval of several hours, for example, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour; and in space to a single institution.
  • Extracorporeal is used in contradistinction to ex vivo which, as used herein, includes more extensive manipulation including extended periods of cell culture and expansion, and/or refrigerated or cryogenic storage or shipment, over several days or longer. [0035] As used herein “expansion” refers to proliferation of cells increasing their number.
  • Activating agents can be used to stimulate proliferation (among other metabolic changes) but can also result in activation-induced death upon initial exposure so that there is no immediate expansion.
  • doubling time can be about 24 hours (which is fairly typical of mammalian cells in vitro generally); in vivo doubling time can be substantially shorter, depending on the presence and type of stimulation. Accordingly, during a limited time of extracorporeal manipulation, even when activating agents are used, such protocols will be effectively expansion-less.
  • an “exogenous protein” refers to a synthetic, recombinant, or other peptide or protein that is not produced by a wild-type cell of that type or is expressed at a lower level in a wild-type cell than in a cell containing the exogenous polypeptide, or that is administered to a subject rather than being produced inside the subject’s body.
  • an exogenous peptide is a peptide or protein encoded by a nucleic acid that was introduced into the cell, which nucleic acid is optionally not retained by the cell.
  • transfection refers to the introduction of nucleic acids into cells by non-viral methods. Transfection can be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation, and lipid-based reagents. In preferred embodiments disclosed herein transfection is mediated by solid lipid nanoparticles (LNP) including targeted LNP (tLNP) (which can also be used to deliver non-nucleic acid payloads into cells).
  • LNP solid lipid nanoparticles
  • tLNP targeted LNP
  • T lymphocytes and natural killer (NK) cells can be reprogrammed with a TCR, a CAR, or an immune cell engager, while a CAR or an immune cell engager are typically used in reprogramming monocytes.
  • stem cells for example hematopoietic stem cells (HSC) or mesenchymal stem cells (MSC)
  • reprogramming refers to correction or amelioration of a genetic defect (for example, a hemoglobinopathy) so that the modified or corrected gene and gene product are the reprogramming agents. Reprogramming can be transient or durable depending on the nature of the engineering agent.
  • Engineerering agent refers to agents that confer expression of a reprogramming agent by a cell, such as an immune cell, particularly a non-B lymphocyte or monocyte.
  • Engineering agents can include nucleic acid molecules, including mRNA, that encode a reprogramming agent.
  • Engineering agents can also include nucleic acid molecules that are or encode components of gene editing systems such as RNA-guided nucleases, guide RNA, and nucleic acid templates for knocking-in a reprogramming agent or knocking- out an endogenous antigen receptor.
  • Gene editing systems comprise base-editors, prime- editors or gene-writers.
  • RNA-guided nucleases include CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, and CasX.
  • CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, and CasX.
  • an mRNA encoding the reprogramming agent can be used as the engineering agent.
  • the engineering agent can comprise mRNA-encoded RNA-directed nucleases, guide RNAs, nucleic acid templates and other components of gene/genome editing systems.
  • gRNA guide RNA
  • sgRNA single guide RNA
  • pegRNA prime editing guide RNA
  • CRISPR clustered regularly interspaced short palindromic repeat
  • tracrRNA trans-activating clustered regularly interspaced short palindromic repeat
  • DNA molecule to be inserted or serve as a template for double-strand break (DSB) repair at a specific genomic locus gRNA
  • sgRNA single guide RNA
  • pegRNA prime editing guide RNA
  • CRISPR clustered regularly interspaced short palindromic repeat
  • tracrRNA trans-activating clustered regularly interspaced short palindromic repeat
  • DSB double-strand break
  • Genome-, gene-, and base- editing technology are reviewed in Anzalone et al., Nature Biotechnology 38:824-844, 2020, Sakuma, Gene and Genome Editing 3-4:100017, 2022, and Zhou et al., MedComm 3(3):e155, 2022, each of which is incorporated by reference for all that they teach about the components and uses of this technology to the extent that it does not conflict with this disclosure.
  • Conditioning agent refers to a biological response modifier (BRM) that enhances the efficiency of engineering an immune cell, expands the number of immune cells available to be engineered or the number of engineered cells in a target tissue (for example, a tumor, fibrotic tissue, or tissue undergoing autoimmune attack), promotes activity of the engineered cell in a target tissue, or broadens the range of operative mechanisms contributing to a therapeutic immune reaction.
  • a conditioning agent can be provided by delivering an encoding nucleic acid in a LNP or tLNP.
  • Exemplary BRMs include cytokines, such as IL-7, IL-15, or IL-18.
  • Conditioning agents can be provided as the agent itself or, when the conditioning agent is a peptide or protein, as a nucleic acid molecule encoding the conditioning agent.
  • “Immune cell,” as used herein, can refer to any cell of the immune system. However, particular aspects can exclude polymorphonuclear leukocytes and/or B cells, or be limited to non-B lymphocytes such as T cell and/or NK cells, or to monocytes such as dendritic cells and/or macrophages in their various forms.
  • lipid nanoparticle means a solid particle, as distinct from a liposome having an aqueous lumen.
  • a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof that is capable of specifically binding to a target or multiple targets.
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest.
  • Exemplary binding moieties of this disclosure include an antibody, a Fab ⁇ , F(ab ⁇ ) 2 , Fab, Fv, rIgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, nanobody, receptor ectodomains or ligand-binding portions thereof, or ligands (e.g., cytokines, chemokines).
  • a “Fab” fragment antigen binding
  • a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand.
  • a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders.
  • assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, and Biacore® analysis.
  • a binding moiety such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.
  • antibody refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen; so-called complementarity determining regions (CDRs).
  • antibody can thus refer to intact or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (camels, alpacas, llamas, etc.) produce antibodies with both the canonical structure and antibodies comprising only heavy chains. The variable region of the camelid heavy chain only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody.
  • Antigen binding fragments and constructs of antibodies include F(ab) 2 , F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH.
  • Such elements can be combined to produce bi- and multi-specific reagents, such as T-cell engagers (for example, Bispecific T-Cell Engagers (BiTEs)) or other immune cell engagers.
  • T-cell engagers for example, Bispecific T-Cell Engagers (BiTEs)
  • BiTEs Bispecific T-Cell Engagers
  • Antibodies can be obtained through immunization, selection from a na ⁇ ve or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that could be used as binding moieties are known in the art.
  • An antibody or other binding moiety “specifically binds” a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M -1 , while not significantly binding other components present in a test sample.
  • Binding domains (or fusion proteins thereof) can be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof).
  • “High affinity” binding domains refer to those binding domains with a Ka of at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 , preferably at least 10 8 M -1 or at least 10 9 M -1 .
  • “Low affinity” binding domains refer to those binding domains with a Ka of up to 10 8 M -1 , up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 .
  • affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 -5 M to 10 -13 M).
  • binding domain polypeptides and fusion proteins can be readily determined using conventional techniques (see, e.g., Scatchard et al., 1949, Ann. N.Y. Acad. Sci.51:660; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent).
  • payload refers to a negatively charged agent that can interact with cationic lipids, such as the ionizable cationic lipids of this disclosure, to become encapsulated within lipid nanoparticles comprising the cationic lipid.
  • the negatively charged agent can be a biologically active small organic molecule, or a macromolecule such as a nucleic acid molecule, a carbohydrate, a peptide, or a polypeptide.
  • a payload can be one or more nucleic acid molecules, RNA or DNA, including mRNA and guide RNA (gRNA) molecules.
  • gRNA guide RNA
  • a payload comprises an mRNA that encodes a biologically active molecule, such as a chimeric antigen receptor, a T cell receptor, an immune cell engager, or the like.
  • biologically active agent refers to any substance that affects a metabolic or physiologic response in a living organism, cells, or cultured cells thereof, including genetically reprograming, permanently or transiently, such organism or cells.
  • therapeutic agent is a substance, or a component of a combination of substances, the biological activity of which can potentially cure, ameliorate, stabilize, prevent, or otherwise beneficially impact a disease, pathological condition, or other disorder.
  • chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, and the like).
  • alkyl generally refers to a monovalent radical (e.g. CH 3 -CH 2 -)
  • a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH 2 -CH 2 -), which is equivalent to the term “alkylene.”
  • aryl refers to the corresponding divalent moiety, arylene.
  • All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for nitrogen, 2 for oxygen, and 2, 4, or 6 for sulfur, depending on the oxidation state of the sulfur atom).
  • alkyl refers to saturated straight and branched chain aliphatic groups having from 1 to 12 carbon atoms. As such, “alkyl” encompasses C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 and C 12 groups.
  • alkenyl as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms.
  • alkenyl encompasses C 2 , C 3 , C 4 , C 5 , C 6 , C 7 , C 8 , C 9 , C 10 , C 11 and C 12 groups.
  • the hydrocarbon chain is unsubstituted.
  • one or more hydrogens of the alkyl or alkenyl group can be substituted with the same or different substituents.
  • Alkynoic refers to a carboxylic acid moiety comprising one or more carbon-carbon triple bonds.
  • hydrogens are unsubstituted.
  • one or more hydrogens of the alkynoic group can be substituted with the same or different substituents.
  • Amide refers to a carboxylic acid derivative comprising a carbonyl group of a carboxylic acid bonded to an amine moiety.
  • Head group refers to the hydrophilic or polar portion of a lipid.
  • Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group.
  • sterols include, without limitation, cholesterol, ergosterol, ⁇ -sitosterol, stigmasterol, stigmastanol, 20-hydroxycholesterol, 22-hydroxycholesterol, and the like.
  • the bond represented as a solid wedge extends above the plane and the bond represented as a dashed wedge extends below the plane when depicting absolute stereochemistry, throughout.
  • the position of X can be referred to as the central branch point and the position of each W can be referred to as a distal branch point.
  • Each of the disclosed ionizable cationic lipids has two tail groups extending from the central branch point and each tail group branches again at its distal branch point.
  • a subscript has a value of “0”
  • the group is absent and the adjoining atoms are bounded to each other. For example, when A 6 is (CH 2 ) 0 , A 6 is absent.
  • a 1 is CH 2
  • a 3 is (CH 2 ) 2-5
  • X is N
  • a 5 is O
  • S NH
  • a 6 is (CH 2 ) 1-2
  • a 7 is (CH 2 ) 1-4 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 2-5
  • X is N
  • a 5 is O
  • a 6 is (CH 2 ) 1-2
  • a 7 is (CH 2 ) 1-4 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-4
  • X is CH
  • a 4 is CH 2
  • a 6 is O, NH, NCH 3 , or CH 2
  • a 7 is (CH 2 ) 0-6 , wherein if A 6 is O, NH, NCH 3 , A 7 is (CH 2 ) 2-4 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-4
  • X is CH
  • a 4 is NH
  • a 6 is O
  • NH NCH 3
  • a 7 is (CH 2 ) 0-6 , wherein if A 6 is O, NH, NCH 3 , A 7 is (CH 2 ) 2-4 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-4
  • X is CH
  • a 4 is NH
  • a 6 is O
  • a 7 is (CH 2 ) 2-4 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-4
  • X is CH
  • a 4 is NCH 3
  • a 6 is O, NH, NCH 3 , or CH 2
  • a 7 is (CH 2 ) 0-6 , wherein if A 6 is O, NH, NCH 3 , A 7 is (CH 2 ) 2-4 .
  • a 1 is (CH 2 ) 2
  • a 3 is (CH 2 ) 1-4
  • X is C-CH 3
  • a 5 is O
  • a 6 is (CH 2 ) 1-2
  • a 7 is (CH 2 ) 1-4 .
  • the number of contiguous connective O A 3 A 4 A 6 atoms present in a span A1 A2 X A 5 A7 i s in the range from 7-17.
  • the number of contiguous connective atoms present in a span O A 3 A 4 A 6 A 1 A 2 X A 5 A7 i s in the range of 7-11 or 7-10.
  • the O 3 4 6 number of contiguous connective atoms present in a span A1 A A A 2 X A 5 A A 7 i s in the range of 10-17 (e.g., in the range of 10-16, or 10-14, or 10-12).
  • the number of contiguous connective atoms present in a span O A 3 4 6 A 1 A 2 A X A 5 A A 7 i s 10.
  • the number of O A 3 A 4 A 6 contiguous connective atoms present in a span A1 A2 X A 5 A7 i s 7.
  • the present inventors have found that changing the number of contiguous connective atoms present in each span can allow for tuning of the pKa of the cationic lipid.
  • Different constituents for Y, A 6 , A 7 , and R 1 allow for tuning of cLogD and c-pKa to achieve a target value of measured pKa within a LNP or a tLNP.
  • CH2 groups in R 1 will tend to increase the lipophilicity (cLogD) of the lipid which in turn will tend to decrease measured pKa of the LNP or tLNP.
  • CH20-3 CH 3 Z N In some embodiments of formula M5, Y is (CH2)0-3 CH 3 and Z is a bond. In ( CH2)0-1 CH 3 Z N some embodiments of formula M5, Y is (CH2)0-1 CH 3 and Z is a bond. For example, in some embodiments of formula M5, Y is and Z is a bond. ( CH2)2-4 OCH 3 Z N [0071] In some embodiments of formula M5, Y is (CH2)0-1 CH 3 and Z is a bond. For example, on some embodiments of formula bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • Y is (CH2)2-4 OCH 3 and Z is a bond.
  • each R 1 is independently C 7 -C 11 alkyl or C 7 -C 11 alkenyl. In some embodiments of formula M5, each R 1 is independently C 7 -C 11 alkyl, e.g., C 7 -C 10 alkyl, or C 7 -C 9 alkyl.
  • each R 1 is independently a linear C 7 -C 11 alkyl, e.g., a linear C 7 -C 10 alkyl, or a linear C 7 -C 9 alkyl.
  • each R 1 is independently (CH 2 ) 6-8 CH 3 . In some of these and other embodiments, R 1 is (CH 2 ) 7 CH 3 .
  • each R 1 is independently a linear C 7 -C 11 alkenyl, e.g., a linear C 7 -C 10 alkenyl, or a linear C 7 -C 9 alkenyl.
  • each R 1 is a linear C 8 alkenyl.
  • each R 1 is independently a branched C 7 -C 11 alkyl, e.g., C 7 -C 10 alkyl, or C 7 -C 9 alkyl.
  • each R 1 is a branched C 8 alkyl.
  • each R 1 is independently a branched C 7 -C 11 alkenyl, e.g., C 7 - C 10 alkenyl, or C 7 -C 9 alkenyl.
  • each R 1 is a branched C 8 alkenyl.
  • each R 1 is the same.
  • each R 1 nearest a common branch point is the same, but those nearest a first common branch point differ from those nearest a second common branch point.
  • each R 1 nearest a common branch point is different but the pair of R1 groups nearest a first common branch point is the same the pair nearest a second common branch point.
  • each W is individually CH or N.
  • each W is CH.
  • each W is N.
  • one W is CH and the other W is N.
  • ionizable cationic lipids that avoid formation of formaldehyde upon degradation. Accordingly, in certain embodiments as described herein, the combination of a R 3 at a beta position of W being O and W being N is avoided. For example, in other certain embodiments, when at least one R 3 at a beta position of a W is O, that W is CH.
  • the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-1: wherein A 1 , A 2 , A 3 , X, A 4 , A 5 , A 6 , A 7 , Y, and R 1 are as otherwise described herein.
  • the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-2: wherein A 1 , A 2 , A 3 , X, A 4 , A 5 , A 6 , A 7 , Y, and R 1 are as otherwise described herein.
  • the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-3: wherein A 1 , A 2 , A 3 , X, A 4 , A 5 , A 6 , A 7 , Y, W, and R 1 are as otherwise described herein.
  • the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-4: wherein A 1 , A 2 , A 3 , X, A 4 , A 5 , A 6 , A 7 , Y, W, and R 1 are as otherwise described herein.
  • each R 1 is independently a C 7 -C 11 alkyl or a C 7 -C 11 alkenyl
  • a 1 is (CH 2 ) 1-2
  • a 2 is O
  • a 3 is (CH 2 ) 1-5
  • a 3 is not CH 2 is X is N
  • X is N, CH, or C-CH 3
  • a 6 is O, S, NH, NCH 3 or (CH 2 ) 0-2
  • a 7 is (CH 2 ) 0-6 , wherein
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-4
  • X is CH
  • a 4 is NH
  • a 6 is O
  • NH NCH 3
  • a 7 is (CH 2 ) 0-6 , wherein if A 6 is O, NH, NCH 3 , A 7 is (CH 2 ) 2-4 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-4
  • X is CH
  • a 4 is NH
  • a 6 is O
  • a 7 is (CH 2 ) 2-4 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-4
  • X is CH
  • a 4 is NCH 3
  • a 6 is O, NH, NCH 3 , or CH 2
  • a 7 is (CH 2 ) 0-6 , wherein if A 6 is O, NH, NCH 3 , A 7 is (CH 2 ) 2-4 .
  • a 1 is (CH 2 ) 2
  • a 3 is (CH 2 ) 1-4
  • X is C-CH 3
  • a 5 is O
  • a 6 is (CH 2 ) 1-2
  • a 7 is (CH 2 ) 1-4 .
  • the number of contiguous connective O A 3 A 4 A 6 atoms present in a span A1 A2 X
  • the number of contiguous connective atoms present in a span: O is in the range of 7-11 or 7-10.
  • the number O of contiguous connective atoms present in a span is in the range of 10-17 (e.g., in the range of 10-16, or 10-14, or 10-12).
  • the number of contiguous connective atoms present in a span O A 3 A 4 A 6 A 1 A 2 X A 5 A7 i s 10.
  • the number of contiguous connective atoms present in a span is 7.
  • changing the number of contiguous connective atoms present in each span can allow for tuning of the pKa of the cationic lipid.
  • CH20-3 CH 3 Z N In some embodiments of formula M4, Y is (CH2)0-3 CH 3 and Z is a bond. In ( CH2)0-1 CH 3 Z N some embodiments of formula M4, Y is (CH2)0-1 CH 3 and Z is a bond. For example, in Z N some embodiments of formula M4, Y is and Z is a bond. ( CH2)2-4 OCH 3 Z N [00111] In some embodiments of formula M4, Y is (CH2)0-1 CH 3 and Z is a bond.
  • Y is and Z is a bond.
  • Y is and Z is a bond.
  • [00127] In some embodiments of formula bond.
  • [00128] In some embodiments of formula bond.
  • [00129] In some embodiments of formula [00132] As described above, in some embodiments of formula M4, it can be desirable to decrease the basicity of the ionizable cationic lipid by selecting an appropriate Y group as Z N N (CH2)0-3CH3 described herein. One such Y group is .
  • a 7 is (CH 2 ) 0
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-5
  • X is CH
  • a 4 is CH 2
  • a 6 is (CH 2 ) 0
  • a 7 is (CH 2 ) 0
  • Y is .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-5
  • X is CH
  • a 4 is O
  • a 6 is (CH 2 ) 0
  • a 7 is (CH 2 ) 0
  • Y is Z N N (CH2)0-3CH3 .
  • a 1 is CH 2
  • a 3 is (CH 2 ) 1-5
  • X is [00134]
  • a 1 is (CH 2 ) 2
  • a 3 is (CH 2 ) 1-5
  • X is CCH 3
  • a 4 is Z N
  • a 5 is absent
  • a 7 is (CH 2 ) 0
  • Y is .
  • each R 1 is independently C 7 -C 11 alkyl or C 7 -C 11 alkenyl.
  • each R 1 is independently C 7 -C 11 alkyl, e.g., C 7 -C 10 alkyl, or C 7 -C 9 alkyl. In certain embodiments of formula M4, each R 1 is independently a linear C 7 -C 11 alkyl, e.g., a linear C 7 -C 10 alkyl, or a linear C 7 -C 9 alkyl. In some embodiments of formula M4 as described herein, each R 1 is independently (CH 2 ) 6-8 CH 3 . In some of these and other embodiments of formula M4, R1 is (CH 2 ) 7 CH 3 .
  • each R 1 is independently a linear C 7 -C 11 alkenyl, e.g., a linear C 7 -C 10 alkenyl, or a linear C 7 -C 9 alkenyl.
  • each R 1 is a linear C 8 alkenyl.
  • each R 1 is independently a branched C 7 -C 11 alkyl, e.g., C 7 -C 10 alkyl, or C 7 -C 9 alkyl.
  • each R 1 is a branched C 8 alkyl.
  • each R 1 is independently a branched C 7 -C 11 alkenyl, e.g., C 7 -C 10 alkenyl, or C 7 -C 9 alkenyl.
  • each R 1 is a branched C 8 alkenyl.
  • the branch point is positioned so that ester carbonyls are not in an ⁇ position relative to the branch point, for example they are in a ⁇ position relative to the branch point.
  • each R 1 is the same.
  • each R 1 nearest a common branch point is the same, but those nearest a first common branch point differ from those nearest a second common branch point. In certain embodiments of formula M4, each R 1 nearest a common branch point is different but the pair of R1 groups nearest a first common branch point is the same the pair nearest a second common branch point.
  • W is CH or N.
  • Z is CH. In certain other embodiments of formula M4, Z is N.
  • Ionizable cationic lipids as described herein, can be useful as a component of lipid nanoparticles for delivering nucleic acids, including DNA, mRNA, or siRNA into cells.
  • the ionizable cationic lipids can have a c-pKa (calculated pKa) in the range of from about 6, 7, or 8 to about 9, 10, or 11.
  • the ionizable cationic lipids have a c-pKa ranging from about 6 to about 10, about 7 to about 10, about 8 to about 10, about 8 to about 9, 6 to 10, 7 to 10, 8 to 10, or 8 to 9.
  • the ionizable cationic lipids have a c-pKa ranging from about 8.4 to about 8.7 or 8.4 to 8.7.
  • the ionizable cationic lipids as described herein can have cLogD ranging from about 9 to about 18, for example, ranging from about 10 to about 18, or about 10 to about 16, to about 10 to about 14, or about 11 to about 18, or about 11 to about 15, or about 11 to about 14, or about 12 to about 14.
  • the ionizable cationic lipids as described herein can have cLogD ranging from 9 to 18, for example, ranging from 10 to 18, or 10 to 16, to 10 to 14, or 11 to 18, or 11 to 15, or 11 to 14, or 12 to 14.
  • the ionizable cationic lipids have a cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4.
  • the ionizable cationic lipids as described herein can have a c-pKa ranging from about 8 to about 11 or from 8 to 11 and a cLogD ranging from about 9 to about 18 or from 9 to 18.
  • the ionizable cationic lipids have a c- pKa ranging from about 8.4 to about 8.7 or from 8.4 to 8.7 and cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4.
  • These ranges can lead to a measured pKa in the LNP ranging from about 6 to about 7 or from 6 to 7, which facilitates ionization in an endosome after delivery into a cell.
  • Ionizable cationic lipids of this disclosure have a branched structure to give the lipid a conical rather than cylindrical shape and such structure helps promote endosomolytic activity when incorporated into an LNP. The greater the endosomolytic activity, the more efficient is release of the biologically active payload (e.g., one or more species of nucleic acid molecules).
  • Another consideration in designing or selecting a particular ionizable cationic lipid for use in an LNP for delivering nucleic acids (or other negatively charged payloads) into cells is the type of cell and, for in vivo delivery, locus within the body targeted for delivery. This is also influenced by pKa. Zhang et al.
  • ionizable lipids with pKa across a wide range can be useful for delivery to various tissues. Of course, other factors can also impact what tissue or cells an LNP will deliver its payload to, including the use of a targeting moiety attached to the surface of the LNP.
  • somewhat greater basicity can be desirable and can be obtained from ionizable cationic lipids with c-pKa and cLogD in the ranges disclosed herein.
  • cLogD of ionizable cationic lipids of this disclosure is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or in a range bound by any pair of these values.
  • Lipid design also accounts for potential biodegradability pathways of target lipids, such as by way of esterases in plasma, liver and other tissues. Another consideration in lipid design is the fate of fragments of ionizable lipids resulting from degradation, such as after esterase cleavage(s). Preferably, the resulting fragments are rapidly cleared from the body without the need for hepatic oxidative metabolism.
  • cLogD is a calculated measure of lipophilicity that accounts for the state of ionization of the lipid molecule at a particular pH, which is a predictor of partitioning of the lipid between water and octanol as a function of pH. More specifically, cLogD is calculated at a specified pH based on cLogP and c-pKa. (LogP is the partition coefficient of a lipid molecule between aqueous (e.g., water) and lipophilic (e.g., octanol) phases). Numerous software packages are available to calculate cLogD values.
  • ionizable cationic lipids of this disclosure having a structure of formula M4, as R 1 increases from C 6 -C 10 , the increase overall lipophilicity of the ionizable cationic lipid will increase, as represented by cLogD.
  • cLogD cLogD
  • c-pKa cLogD and c-pKa values within the desired range(s), as described herein.
  • Specific cLogD and c-pKa values have been calculated using ACD Labs Structure Designer v 12.0 for ionic cationic lipids of the disclosure.
  • cLogP was calculated using ACD Labs Version B; cLogD was calculated at pH 7.4.
  • the ionizable cationic lipid has the structure CICL-250: CICL-250 [00144] In some embodiments, the ionizable cationic lipid has the structure CICL-291: [00145] In some embodiments, the ionizable cationic lipid has the structure CICL-292: [00146] In some embodiments, the ionizable cationic lipid has the structure CICL-293: [00147] In some embodiments, the ionizable cationic lipid has the structure CICL-294: [00148] In some embodiments, the ionizable cationic lipid has the structure CICL-295: [00149] In some embodiments, the ionizable cationic lipid has the structure CICL-295: [00149] In some embodiments, the ionizable cationic lipid has the structure CICL-295: [00149] In some embodiments, the ionizable cationic lipid has the structure CICL
  • R 4 is H.
  • R 4 is a protecting group (i.e., PG 1 ).
  • PG 1 can be selected from base labile or acid labile protecting groups as known in the art.
  • R 4 is an acid labile protecting group such as t-butoxycarbonyl (BOC) or benzyloxycarbonyl (Cbz).
  • R 4 is a base labile protecting group such as a trimethylsilylethoxycarbonyl moiety.
  • R 5 is .
  • R 5 is .
  • the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M5 has the structure of formula I5-3:
  • R 6 is .
  • a 1 is CH 2 or CH 2 CH 2
  • a 3 is CH 2 or CH 2 CH 2
  • X is C-CH 3 .
  • a 1 is CH 2
  • a 3 is CH 2 CH 2
  • X is C-CH 3
  • R 6 is OH.
  • a 1 is CH 2 or CH 2 CH 2
  • a 3 is CH 2 or CH 2 CH 2
  • X is N.
  • a 1 is CH 2
  • a 2 is O
  • a 3 is CH 2 CH 2
  • X is N
  • R 4 is a protecting group (i.e., PG 1 ).
  • PG 1 can be selected from base labile or acid labile protecting groups as known in the art.
  • R 4 is an acid labile protecting group such as t-butoxycarbonyl (BOC) or benzyloxycarbonyl (Cbz).
  • R 4 is a base labile protecting group such as a trimethylsilylethoxycarbonyl moiety.
  • a 1 and A 3 are CH 2
  • X is CH
  • a 4 is CH 2 , NH, NCH 3 , or O.
  • the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M4 has the structure of formula I4-2: wherein each R 1 is independently a C 7 -C 11 alkyl or a C 7 -C 11 alkenyl; each W is independently CH or N; A 1 is CH 2 or CH 2 CH 2 , A 2 is O, A 3 is CH 2 or CH 2 CH 2 , wherein A 3 is not CH 2 if X is N, X is N, CH, or C-CH 3 , and A 4 is CH 2 , NH, NCH 3 , or O, and [00202] In various embodiments of formula I4-2, R 5 is OH.
  • R 5 is .
  • a 1 and A 3 are CH 2 , then X is CH, A 4 is CH 2 , NH, NCH 3 , or O.
  • a 1 and A 3 are CH 2 , X is CH, A 4 is NH 2 , and
  • a 1 and A 3 are CH 2 , X is CH, A 4 is CH 2 , and R 5 is OH.
  • the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M4 has the structure of formula I4-3: wherein each R 1 is independently a C 7 -C 11 alkyl or a C 7 -C 11 alkenyl; each W is independently CH or N; A 1 is CH 2 or CH 2 CH 2 , A 2 is O, A 3 is CH 2 or CH 2 CH 2 , wherein A 3 is not CH 2 if X is N, and X is N, CH, or C-CH 3 ; and [00208] In various embodiments of formula I4-3, R 6 is OH. [00209] In various embodiments of formula I4-3, R 6 is .
  • a 1 is CH 2 or CH 2 CH 2
  • a 3 is CH 2 or CH 2 CH 2
  • X is C-CH 3
  • a 1 is CH 2
  • a 3 is CH 2 CH 2
  • X is C-CH 3
  • R 6 is OH.
  • a 1 is CH 2 or CH 2 CH 2
  • a 3 is CH 2 or CH 2 CH 2
  • X is N.
  • the fatty acid tails are designed to comprise esters in a position that minimizes steric hindrance of ester cleavage. For example, while a single fatty acid tail will tend to extend away from the ester carbonyl to provide the most energetically favorable position, the presence of two tails leads to the tails extending in opposite directions to provide the most energetically favorable conformation. Conformation is dynamic and the fatty acid tails can also move through less energetically favorable positions.
  • one of the tails can extend toward the carbonyl and sterically hinders cleavage of the ester. Accordingly, large branches immediately adjacent to the ester carbonyl were avoided in the design of the ionizable cationic lipids disclosed herein.
  • esters i.e., the esters nearest to the central branchpoint nitrogen atom
  • the transesterification would be a simple intramolecular ester exchange and no piece of the molecule would be released. Accordingly, to promote biodegradation, it is advantageous for a methylene to be at an alpha position of W.
  • An advantage of ionizable cationic lipids of this disclosure is that, at least in part, the toxicity associated with quaternary ammonium cationic lipids can be avoided.
  • the use of ionizable cationic lipids of this disclosure in an LNP obviates the need for quaternary ammonium cationic lipids and, thereby, can mitigate or avoid potential LNP toxicity.
  • use of an LNP or tLNP of this disclosure causes no detectable toxicity to cells or in a subject.
  • use of an LNP or tLNP of this disclosure causes no more than mild toxicity to cells or in a subject that is asymptomatic or induces only mild symptoms that do not require intervention. In certain embodiments, use of an LNP or tLNP of this disclosure causes no more than moderate toxicity to cells or in a subject which may impair activities of daily living that requires only minimal, local, or non-invasive interventions.
  • the relationship between the efficacy and toxicity of a drug is generally expressed in terms of therapeutic window and therapeutic index. Therapeutic window is the dose range from the lowest dose that exhibits a detectable therapeutic effect up to the maximum tolerated dose (MTD); the highest dose that will the desired therapeutic effect without producing unacceptable toxicity.
  • MTD maximum tolerated dose
  • therapeutic index is calculated as the ratio of LD 50 :ED 50 when based on animal studies and TD 50 :ED 50 when based on studies in humans (though this calculation could also be derived from animal studies and is sometimes called the protective index), where LD 50 , TD 50 , and ED 50 are the doses that are lethal, toxic, and effective in 50% of the tested population, respectively.
  • LD 50 , TD 50 , and ED 50 are the doses that are lethal, toxic, and effective in 50% of the tested population, respectively.
  • Toxicities and adverse events can be graded according to a 5-point scale. A grade 1 or mild toxicity is asymptomatic or induces only mild symptoms; may be characterized by clinical or diagnostic observations only; and intervention is not indicated.
  • a grade 2 or moderate toxicity may impair activities of daily living (such as preparing meals, shopping, managing money, using the telephone, etc.) but only minimal, local, or non- invasive interventions are indicated.
  • Grade 3 toxicities are medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization is indicated; activities of daily living related to self-care (such as bathing, dressing and undressing, feeding oneself, using the toilet, taking medications, and not being bedridden) may be impaired.
  • Grade 4 toxicities are life-threatening and urgent intervention is indicated.
  • Grade 5 toxicity produces an adverse event-related death.
  • a toxicity is confined to grade 2 or less, grade 1 or less, or produces no observed toxicity.
  • LNPs deliver primarily to the liver. Liver toxicity has been the major dose- limiting parameter observed with LNP-containing pharmaceuticals.
  • ONPATTRO® comprising the ionizable lipid MC3
  • NOAEL no observed adverse effect level
  • a benchmark LNP comprising the ionizable cationic lipid ALC-0315, used in the SARS-CoV-2 vaccine COMIRNATY®, caused elevated levels of liver enzymes and acute phase proteins at single doses of ⁇ 1 mg/kg in the rat.
  • this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M5 (e.g., CICL-309, CICL-310, and CICL-311).
  • this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M4 (e.g., CICL-250, CICL-291, CICL-292, CICL-293, CICL-294, CICL-295, CICL-296, CICL-297, CICL-298, CICL-299, CILC-300, CICL-301, CICL-302, and CICL-303).
  • Table 2 provides a summary of substituents in formula M4 of CICL-250 and CICL-291 – CICL-303.
  • this disclosure provides a method of synthesizing an ionizable cationic lipid of formula CICL-250, CICL-297, CICL-291, and CICL-298 comprising the synthesis step as shown in Scheme 1, wherein the methylated acylimidazolide is reacted with HB in the presence of a mild base such as Et 3 N or Me 3 N and HB is an alcohol, amine, or thiol,, and the rest of the substituents are defined the same as in formulas CICL- 250, CICL-297, CILC-291, and CICL-298.
  • a mild base such as Et 3 N or Me 3 N
  • HB is an alcohol, amine, or thiol
  • the synthesis step shown in Scheme 1 comprises reacting a diester-X-amine (1- A or 1B) with 1,1'-carbonyldiimidazole (CDI) to provide an imidazolecarboxyamide (1-C or 1- D); and after activation of the imidazolecarboxyamide (1-C or 1-D) it is coupled with a desired B (i.e. HB) to provide 1-E, wherein B is A 5 -A 6 -A 7 -Y, or 1-F, wherein B is A 6 -A 7 -Y.
  • CDI 1,1'-carbonyldiimidazole
  • the imidazolecarboxyamide 1-C or 1-D synthesis is carried out in an organic solvent (e.g., without limitation, CH 2 Cl 2 ) in the presence of a basic catalyst (e.g., without limitation, Et 3 N).
  • the synthesis can comprise first reacting the imidazolecarboxyamide 1-C or 1-D with MeOTf, then reacting with the desired B (i.e. HB) in the presence of a base (e.g., trimethylamine). The reaction can be carried out in an organic solvent (e.g., acetonitrile).
  • this disclosure provides a method of synthesizing an ionizable cationic lipid of formula CICL-292 and CICL-299 comprising the synthesis step shown in Scheme 2, all substituents are defined the same as in formula CICL-292 or CICL 299.
  • Scheme 2 [00230] The synthesis step shown in Scheme 2 comprises coupling a diester-X-A 5 -acid 2- A with an amine/alcohol (i.e. HB) to provide an amide/ester having the structure of formula 2B, wherein B is A 6 -A 7 -Y.
  • the coupling reaction is carried out in an organic solvent (e.g., THF) in the presence of basic catalysts (e.g., EDC-HCl and DMAP, or HATU and i-Pr 2 NEt).
  • basic catalysts e.g., EDC-HCl and DMAP, or HATU and i-Pr 2 NEt.
  • the synthesis step shown in Scheme 3 comprises coupling the diester-X- amine/alcohol (3A or 3B) with a desired base, HOOC-B or HO-B, to provide an amide/ester having the structure of formula 3-C, wherein B is A 6 -A 7 -Y, or 3-D, wherein B is A 6 -A 7 -Y.
  • the coupling reaction is carried out in an organic solvent (e.g., acetonitrile, THF, or CH 2 Cl 2 ) in the presence of a nucleophilic catalyst (e.g., DMAP), an acidic catalyst (e.g., EDC-HCl) and a basic catalyst (e.g., Et 3 N).
  • a nucleophilic catalyst e.g., DMAP
  • an acidic catalyst e.g., EDC-HCl
  • a basic catalyst e.g., Et 3 N
  • this disclosure provides a method of synthesizing an ionizable cationic lipid of formula CICL-309, CICL-310, and CICL-311 comprising the synthesis steps as shown in Scheme 4 wherein the methylated acylimidazolide is reacted with HB in the presence of a mild base such as Et 3 N or Me 3 N and HB is an alcohol, amine, or thiol, and the rest of the substituents are defined the same as in formulas CICL-309, CICL-310, and CICL-311.
  • a mild base such as Et 3 N or Me 3 N
  • HB is an alcohol, amine, or thiol
  • the synthesis step shown in Scheme 4 comprises reacting a diester-X-amine (4- A) with 1,1'-carbonyldiimidazole (CDI) to provide an imidazolecarboxyamide (4-C); and after activation of the imidazolecarboxyamide (4-C) it is coupled with a desired B (i.e. HB) to provide 4-E, wherein B is A 5 -A 6 -A 7 -Y.
  • B i.e. HB
  • All substituents are defined the same as those in formula CICL-309, CICL-310, or CICL-311.
  • the imidazolecarboxyamide 4-C synthesis is carried out in an organic solvent (e.g., without limitation, CH 2 Cl 2 ) in the presence of a basic catalyst (e.g., without limitation, Et 3 N).
  • the synthesis can comprise first reacting the imidazolecarboxyamide 4-C with MeOTf, then reacting with the desired B (i.e. HB) in the presence of a base (e.g., trimethylamine).
  • a base e.g., trimethylamine
  • the reaction can be carried out in an organic solvent (e.g., acetonitrile).
  • HB is selected from H-A 6 -A 7 -Y, H-A 5 -A 6 -A 7 -Y, and HOOC-A 6 -A 7 -Y are used under coupling conditions to provide the ionizable cationic lipids of formulas M5 and M4 as described herein.
  • H-A 6 -A 7 -Y is selected from the group consisting of A 6 and A 7 are as otherwise described herein.
  • H-A 6 -A 7 -Y compounds disclosed herein are commercially available, are known in the scientific literature, or can be made using procedures familiar to the person of ordinary skill in the art, provided from commercial sources, or the general procedures described in the Examples below. [00238]
  • H-A 6 -A 7 -Y is selected from any one of [00239]
  • H-A 5 -A 6 -A 7 -Y is selected from the are as otherwise described herein.
  • H-A 5 -A 6 -A 7 -Y compounds disclosed herein are commercially available, are known in the scientific literature, or can be made using procedures familiar to the person of ordinary skill in the art, provided from commercial sources, or the general procedures described in the Examples below.
  • HOOC-A 6 -A 7 -Y is HOOC-A 6 -A 7 -Y compounds disclosed herein are commercially available, are known in the scientific literature, or can be made using procedures familiar to the person of ordinary skill in the art, provided from commercial sources, or the general procedures described in the Examples below.
  • any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins.
  • the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., Still, Kahn, Mitra, J. Org. Chem.1978, 43, 2923-292, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969. [00243] During any of the processes for preparation of the subject compounds, it can be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned.
  • LNPs Lipid Nanoparticles
  • tLNPs Targeted LNPs
  • an LNP comprises an ionizable cationic lipid of formula M4 and/or M5 and a phospholipid, a sterol, a co-lipid, a PEGylated lipid, or a combination thereof.
  • the PEG-lipids are not functionalized PEG-lipids.
  • the PEG-lipids are functionalized PEG- Lipids.
  • the LNP comprises at least one PEG-lipid that is functionalized and at least one PEG-lipid that is not functionalized.
  • this disclosure provides a targeted lipid nanoparticle (tLNP) comprising an ionizable cationic lipid of formula M4 and/or M5.
  • the aforementioned tLNP can further comprise one or more of a phospholipid, a sterol, a co-lipid, and a PEG-lipid, or a combination thereof, and a functionalized PEG-lipid.
  • “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid.
  • the functionalized PEG-lipid can be reacted with a binding moiety (e.g., an antibody or Fab) after the LNP is formed, so that the binding moiety is conjugated to the PEG portion of the lipid.
  • a binding moiety of a LNP or tLNP comprises an antigen binding domain, an antigen, a ligand-binding domain of a receptor, or a receptor ligand.
  • a binding moiety comprises a complete antibody, an F(ab) 2 , an Fab, a minibody, a single-chain Fv (scFv), a diabody, a VH domain, or a nanobody, such as a VHH or single domain antibody.
  • the receptor ligand is a carbohydrate, for example, a carbohydrate comprising terminal galactose or N-acetylgalactosamine units, which are bound by the asialoglycoprotein receptor.
  • These binding moieties constitute means for LNP targeting. Some embodiments specifically include one or more of these binding moieties. Other embodiments specifically exclude one or more of these binding moieties.
  • LNP and tLNP Compositions [00248] The LNP composition contributes to the formation of stable LNPs and tLNPs, efficient encapsulation of a payload, protection of a payload from degradation until it is delivered into a cell, and promotion of endosomal escape of a payload into the cytoplasm.
  • LNP and tLNP compositions are generally disclosed in PCT/US2024/032141, filed 31 May 2024, published as WO2024/249954, and entitled Lipid Nanoparticle Formulations and Compositions, which is incorporated by reference for all that it teaches about the design, formation, characterization, properties, and use of LNPs and tLNPs.
  • the LNPs and/or tLNPs can include the various components in amounts sufficient to provide a nanoparticle with a desired shape, fluidity, and bio-acceptability as described herein.
  • the LNPs comprises at least one ionizable cationic lipid as described herein in an amount in the range of from about 35 to about 65 mol%, e.g., in an amount of from about 40 to about 65 mol%, about 40 to about 60 mol%.
  • the LNP or tLNP comprises about 58 mol% or 62 mol% ionizable cationic lipid.
  • is the LNP (or tLNP) comprises a phospholipid in an amount in the range of from about 7 to about 30 mol%, e.g., in an amount of from about 13 to about 30 mol%.
  • the LNP or tLNP comprises about 10 mol% phospholipid.
  • the LNP (or tLNP) comprises a sterol in an amount in the range of from about 20 to about 50 mol%, e.g., in an amount in the rage of from about 20 to about 45 mol%, or about 30 to about 50 mol%, or about 30 to about 45 mol%.
  • the LNP or tLNP comprises about 30.5, 26.5, or 23.5 mol% sterol.
  • the LNP (or tLNP) comprises at least one co-lipid in an amount in the range of from about 1 to about 30 mol%.
  • the LNP (or tLNP) comprises at least one unfunctionalized PEG-lipid in an amount of from 0 to about 5 mol%, e.g., in the range of amount 0 to about 3 mol%, or about 0.1 to about 5 mol%, or about 0.5 to about 5 mol%, or about 0.5 to about 3 mol%. In some embodiments, the LNP or tLNP comprises about 1.4 mol% unfunctionalized PEG-lipid. In some embodiments, the LNP or tLNP comprises at least one functionalized PEG-lipid in an amount in the range of from about 0.1 to about 5 mol%, e.g., in the range of from about 0.1 to 0.3 mol%.
  • the LNP or tLNP comprises about 0.1 mol% functionalized PEG-lipid. In some embodiments, the functionalized PEG-lipid is conjugated to a binding moiety.
  • this disclosure provides an LNP or tLNP, wherein the LNP or tLNP comprises about 35 mol% to about 65 mol% of an ionizable cationic lipid, about 0.5 mol% to about 3 mol% of a PEG-lipid (including non-functionalized PEG-lipid and optionally a functionalized PEG-lipid), about 7 mol% to about 13 mol% of a phospholipid, and about 30 mol% to about 50 mol% of a sterol.
  • an LNP or tLNP comprises a payload with a net negative charge for example, a peptide, a polypeptide, a protein, a small molecule, or a nucleic acid molecule, and combinations thereof.
  • a payload is generally encompassed by or in the interior of an LNP or tLNP.
  • dosages always refer to the amount of payload being provided.
  • a payload comprises one or more species of nucleic acid molecule.
  • tLNP encapsulating mRNA dosages are typically in the range of 0.05 to 5 mg/kg without regard for recipient species. In some embodiments, the dosage is in the range of 0.1 to 1 mg/kg.
  • the ratio of total lipid to nucleic acid is about 10:1 to about 50:1 on a weight basis. In some embodiments, the ratio of total lipid to nucleic acid is about 10:1, about 20:1, about 30:1, or about 40:1 to about 50:1, or 10:1 to 20:1, 30:1, 40:1 or 50:1, or any range bound by a pair of these ratios.
  • the N/P ratio is from about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 6, about 5 to about 6, or about 6. In some instances, the N/P ratio is from 3 to 9, 3 to 7, 3 to 6, 4 to 6, 5 to 6, or 6.
  • an LNP or tLNP comprises about 40 mol% to about 62 mol% ionizable cationic lipid. In some embodiments, an LNP or tLNP comprises about 1 mol% to about 2 mol% total PEG-lipid.
  • an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol%, for example about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% functionalized PEG-lipid.
  • a binding moiety is conjugated to functionalized PEG-lipid.
  • a tLNP is an LNP that further comprises an antibody (for example, a whole IgG) as the binding moiety which is present at an antibody:mRNA ratio (w/w) of about 0.3 to about 1.0 w/w.
  • the LNP or tLNP has a hydrodynamic diameter of 50 to 150 nm and in some embodiments the hydrodynamic diameter is ⁇ 120, ⁇ 110, ⁇ 100, or ⁇ 90 nm. Uniformity of particle size is also desirable with a polydispersity index (PDI) of ⁇ 0.2 (on a scale of 0 to 1) being acceptable. Both hydrodynamic diameter and polydispersity index are determined by dynamic light scattering (DLS). Particle diameter as assessed from cryo- transmission electron microscopy (Cryo-TEM) can be smaller than the DLS-determined value.
  • Phospholipids [00253] As described above, in various embodiments, the LNPs and tLNPs include a phospholipid.
  • phospholipids are amphiphilic molecules. Due to the amphiphilic nature of phospholipids, these molecules are known to form bilayers and by including them in the LNPs and tLNPs, as described herein, they can provide membrane formation, stability, and rigidity.
  • phospholipids include a hydrophilic head group, including a functionalized phosphate group, and two hydrophobic tail groups derived from fatty acids.
  • the phospholipids include a phosphate group functionalized with ethanolamine, choline, glycerol, serine, or inositol.
  • the phospholipid includes two hydrophobic tail groups derived from fatty acids.
  • These hydrophobic tail groups can be derived from unsaturated or saturated fatty acids.
  • the hydrophobic tail groups can be derived from a C 12 -C 20 fatty acid.
  • a phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3- phosphocholine (DAPC), or a combination thereof.
  • DOPE dioleoylphosphatidyl ethanolamine
  • DMPC dimyristoylphosphatidyl choline
  • DSPC distearoylphosphatidylcholine
  • DMPG dimyristoylphosphatidyl glycerol
  • DPPC dipalmitoyl phosphatidylcholine
  • DAPC 1,2-diarachidoyl-sn-glycero-3- phosphocholine
  • the phospholipid is selected from the group comprising dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2- diarachidoyl-sn-glycero-3-phosphocholine (DAPC).
  • DOPE dioleoylphosphatidyl ethanolamine
  • DMPC dimyristoylphosphatidyl choline
  • DSPC distearoylphosphatidylcholine
  • DMPG dimyristoylphosphatidyl glycerol
  • DPPC dipalmitoyl phosphatidylcholine
  • DAPC 1,2- diarachidoyl-sn-glycero-3-phosphocholine
  • the phospholipid is diste
  • Phospholipids can contribute to formation of a membrane, whether monolayer, bilayer, or multi-layer, surrounding the core of the LNP or tLNP. Additionally, phospholipids such as DSPC, DMPC, DPPC, DAPC impart stability and rigidity to membrane structure. Phospholipids, such as DOPE, impart fusogenicity. Further phospholipids, such as DMPG, which attains negative charge at physiologic pH, facilitates charge modulation. Thus, phospholipids constitute means for facilitating membrane formation, means for imparting membrane stability and rigidity, means for imparting fusogenicity, and means for charge modulation.
  • an LNP or tLNP has about 7 mol% to about 13 mol% phospholipid, about 7 mol% to about 10 mol% phospholipid, or about 10 mol% to about 13 mol% phospholipid. In certain embodiments, an LNP has about 7 mol%, about 10 mol%, or about 13 mol% phospholipid. In certain instances, the phospholipid is DSPC. In certain instances, the phospholipid is DAPC.
  • Sterols [00256] The disclosed LNP and tLNP comprise a sterol. Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group.
  • sterols include, without limitation, cholesterol, ergosterol, ⁇ -sitosterol, stigmasterol, stigmastanol, 20- hydroxycholesterol, 22-hydroxycholesterol, and the like.
  • a sterol is cholesterol, 20-hydroxycholesterol, 22- hydroxycholesterol, or a phytosterol.
  • the phytosterol comprises campesterol, sitosterol, or stigmasterol, or combinations thereof.
  • the cholesterol is not animal-sourced but is obtained by synthesis using a plant sterol as a starting point.
  • LNPs incorporating C-24 alkyl (such as methyl or ethyl) phytosterols have been reported to provide enhanced gene transfection.
  • the length of the alkyl tail, the flexibility of the sterol ring, and polarity related to a retain C-3 -OH group are important to obtaining high transfection efficiency. While ⁇ -sitosterol and stigmasterol performed well, vitamin D2, D3 and calcipotriol, (analogs lacking intact body of cholesterol) and betulin, lupeol ursolic acid and olenolic acid (comprising a 5 th ring) should be avoided.
  • Sterols serve to fill space between other lipids in the LNP or tLNP and influence LNP or tLNP shape. Sterols also control fluidity of lipid compositions, reducing temperature dependence. Thus, sterols such as cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, ⁇ -sitosterol, and stigmasterol constitute means for controlling LNP shape and fluidity or sterol means for increasing transfection efficiency. In designing a lipid composition for a LNP or tLNP, in some embodiments, sterol content can be chosen to compensate for different amounts of other types of lipids, for example, ionizable cationic lipid or phospholipid.
  • an LNP or tLNP has about 27 mol% or about 30 mol% to about 50 mol% sterol, or about 30 mol% to about 38 mol% sterol. In certain embodiments, an LNP or tLNP has about 30.5 mol%, about 33.5 mol%, or about 37.5 mol% sterol. In certain instances, the sterol is cholesterol. In certain embodiments, the sterol is a mixture of sterols, for example, cholesterol and ⁇ -sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, the sterol is about 25 mol% 20-hydroxycholesterol and about 75 mol% cholesterol.
  • the sterol is about 25 mol% ⁇ -sitosterol and about 75 mol% cholesterol. In some instances, the sterol is about 50 mol% ⁇ -sitosterol and about 50 mol% cholesterol. In certain embodiments, an LNP or tLNP has 27 mol% or 30 mol% to 50 mol% sterol or 30 mol% to 38 mol% sterol. In further embodiments, an LNP or tLNP has 30.5 mol%, 33.5 mol%, or 37.5 mol% sterol. In certain instances, a sterol is cholesterol.
  • a sterol is a mixture of sterols, for example, cholesterol and ⁇ -sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, a sterol is 25 mol% 20- hydroxycholesterol and 75 mol% cholesterol. In further instances, a sterol is 25 mol% ⁇ - sitosterol and 75 mol% cholesterol. In still further instances, a sterol is 50 mol% ⁇ -sitosterol and 50 mol% cholesterol. [00258] With respect to LNPs or tLNPs of this disclosure, in some embodiments, a co- lipid is absent or comprises an ionizable lipid, anionic or cationic.
  • a co-lipid can be used to adjust various properties of an LNP or tLNP, such as surface charge, fluidity, rigidity, size, stability, and the like properties.
  • a co-lipid is an ionizable lipid, such as cholesterol hemisuccinate (CHEMS) or an ionizable lipid of this disclosure.
  • CHEMS cholesterol hemisuccinate
  • a co-lipid is a charged lipid, such as a quaternary ammonium head group containing lipid.
  • a quaternary ammonium head group containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3 ⁇ -(N-(N’,N’- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof.
  • these compounds a chloride, bromide, mesylate, or tosylate salt.
  • the co-lipid is a quaternary ammonium head group containing lipid
  • the quaternary ammonium head group containing lipid makes up no more than 50 mol% of the total cationic lipid, for example, from 5 to 50% of the total cationic lipid.
  • an LNP or tLNP were to have cationic lipid content of 70 mol% and 5 to 50 mol% of the total cationic lipid as quaternary ammonium lipid, the LNP or tLNP would have from 3.5 mol% quaternary ammonium lipid and 66.5 mol% ionizable cationic lipid to 35 mol% each of quaternary ammonium lipid and ionizable cationic lipid.
  • the disclosed ionizable lipids of formulas M4 and M5 have a measured pKa ranging from about 6 to about 7 or from 6 to 7, they can contribute substantial endosomal release activity to an LNP or tLNP containing the ionizable lipid. More acidic or basic ionizable lipids of formulas M4 and M5 can contribute surface charge and thus serve as a co-lipid as described immediately above. In such cases, it can be advantageous to incorporate another lipid with fusogenic activity into an LNP or tLNP of this disclosure.
  • a PEG-lipid is a lipid conjugated to a polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG-lipid is a C 14 -C 20 lipid conjugated with a PEG.
  • the PEG-lipid is a C 14 -C 20 lipid conjugated with a PEG, or a C 14 -C 18 lipid conjugated with a PEG, or a C 14 -C 16 lipid conjugated with a PEG.
  • the PEG-lipid is a fatty acid conjugated with a PEG.
  • the fatty acid of the PEG-lipid can have a variety of chain lengths.
  • the PEG-lipid is a fatty acid conjugated with PEG, wherein the fatty acid chain length is in the range of C 14 -C 20 (e.g., in the range of C 14 -C 18 , or C 14 -C 16 ).
  • PEG-lipids with fatty acid chain lengths less than C 14 are too rapidly lost from the (t)LNP while those with chain lengths greater than C 20 are prone to difficulties with formulation.
  • the LNP comprises one or more PEG-lipids and/or functionalized PEG-lipids; when both a functionalized and unfunctionalized PEG-lipid, the PEG-lipid present they can be the same or different; and one or more ionizable cationic lipids; the LNP can further comprise a phospholipid, a sterol, a co-lipid, or any combination thereof.
  • the term “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid.
  • the functionalized PEG-lipid can be reacted with a binding moiety so that the binding moiety is conjugated to the PEG portion of the lipid.
  • the conjugated binding moiety can thus serve as a targeting moiety for the LNP to constitute a tLNP.
  • the binding moiety is conjugated to the functionalized PEG- lipid after an LNP comprising the functionalized PEG-lipid is formed.
  • the binding moiety is conjugated to the PEG-lipid and then the conjugate is inserted into a previously formed LNP.
  • the LNP is a tLNP comprising one or more functionalized PEG-lipids that has been conjugated to a binding moiety.
  • the tLNP also comprises PEG-lipids not functionalized or conjugated with a binding moiety.
  • the functionalization is a maleimide.
  • the functionalization is a bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide moiety at the terminal hydroxyl end of the PEG moiety.
  • the binding moiety comprises an antibody or antigen binding portion thereof.
  • the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group.
  • the conjugation linkage comprises a reaction product of a thiol in the binding moiety with a functionalized PEG-lipid.
  • the functionalization is a maleimide, azide, alkyne, dibenzocyclooctyne (DBCO), bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide.
  • the binding moiety comprises an antibody or antigen binding portion thereof.
  • the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group. [00263]
  • PEG can be made in a large range of sizes.
  • n ⁇ 22 to 113 is used to represent PEG-lipids incorporating PEG moieties in the range of PEG-1000 to PEG-5000 such as PEG-1000, PEG-1500, PEG- 2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000, although some molecules from preparations at the average molecular weight boundaries will have an n outside that range.
  • n ⁇ 22 is used to represent PEG-lipids incorporating PEG moieties from PEG-1000
  • n ⁇ 45 is used to represent PEG-lipids incorporating PEG moieties from PEG-2000
  • n ⁇ 67 is used to represent PEG-lipids incorporating PEG moieties from PEG-3000
  • n ⁇ 90 is used to represent PEG-lipids incorporating PEG moieties from PEG-4000
  • n ⁇ 113 is used to represent PEG-lipids incorporating PEG moieties from PEG-5000.
  • a PEG is of 500-5000 or 1000-5000 Da molecular weight (MW).
  • the PEG of the PEG-lipid has a molecular weight in the range of 1500-5000 Da or 2000-5000 Da.
  • the PEG-lipid has a molecular weight in the range of 500-4000 Da, or 500-3000 Da, or 1000-4000 Da, or 1000-3000, or 1000-2500, or 1500-4000, or 1500-3000, or 1500-2500 Da.
  • the PEG unit has a MW of 2000 Da (sometime abbreviated as PEG(2k)).
  • PEG moieties of PEG-1000, PEG-2000, or PEG- 5000 comprise a DSG-PEG, for example, DSG-PEG-2000.
  • Certain embodiments comprise a DSPE-PEG, for example, DSPE-PEG-2000.
  • Certain embodiments comprise both DSG-PEG-2000 and/or DSPE-PEG2000.
  • the PEG moiety is PEG-500 to PEG-5000 such as PEG- 500, PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000.
  • the PEG moiety is PEG-2000.
  • the PEG unit has a MW of 2000 Da.
  • Common PEG-lipids fall into two classes diacyl glycerols and diacyl phospholipids.
  • diacyl glycerol PEG-lipids include DMG-PEG (1,2-dimyristoyl- glycero-3-methoxypolyethylene glycol), DPG-PEG (1,2-dipalmitoyl-glycero-3- methoxypolyethylene glycol), DSG-PEG (1,2-distearoyl-glycero-3-methoxypolyethylene glycol), and DOG-PEG (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol).
  • diacyl phospholipids examples include DMPE-PEG (1,2-dimyristoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol), DPPE-PEG (1,2-dipalmitoyl-glycero-3-phosphoethanolamine- 3-methoxypolyethylene glycol), DSPE-PEG (1,2-distearoyl-glycero-3-phosphoethanolamine- 3-methoxypolyethylene glycol), and DOPE-PEG (1,2-dioleoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol).
  • the MW2000 PEG-lipid (e.g., a PEG-lipid comprising a PEG of a molecular weight of 2000 Da) comprises DMG-PEG2000 (1,2-dimyristoyl-glycero- 3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3- methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3- methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-
  • the PEG unit has a MW of 2000 Da.
  • the MW2000 PEG-lipid comprises DMrG- PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DPrG-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSrG-PEG2000 (1,2- distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DorG-PEG2000 (1,2-dioleoyl- glycero-3-methoxypolyethylene-rac-glycol-2000), DMPEr-PEG200 (1,2-dimyristoyl-rac- glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPEr-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glyco
  • the glycerol in these lipids is chiral.
  • the PEG-lipid is racemic.
  • optically pure antipodes of the glycerol portion can be employed, that is, the glycerol portion is homochiral.
  • optically pure means ⁇ 95% of a single enantiomer (D or L).
  • the enantiomeric excess is ⁇ 98%.
  • the enantiomeric excess is ⁇ 99%.
  • PEG-lipids including achiral PEG-lipids built on a symmetric dihydroxyacetone scaffold, a symmetric 2-(hydroxymethyl)butane-1,4-diol, or a symmetric glycerol scaffold, are disclosed in U.S. Provisional Application No.63/362,502, filed on April 5, 2022, and PCT/US2023/017648 filed on April 5, 2023 (WO 2023/196445), both entitled PEG-Lipids and Lipid Nanoparticles, which are incorporated by reference in their entirety. [00267] The above examples are presented as methoxypolyethylene glycols, but the terminus need not necessarily be methoxyl.
  • the PEG moiety of the PEG lipids can terminate with a methoxyl, a benzyloxyl, a 4-methoxybenzyloxyl, or a hydroxyl group (that is, an alcohol).
  • the terminal hydroxyl facilitates functionalization.
  • the methoxyl, benzyloxyl, and 4-methoxybenzyloxyl groups are advantageously provided for PEG-lipid that will be used without functionalization as a component of the LNP. However, all four of these alternatives are useful as the (non-functionalized) PEG-lipid component of LNPs.
  • the 4- methoxybenzyloxyl group is readily removed to generate the corresponding hydroxyl group.
  • the 4- methoxybenzyloxyl group offers a convenient path to the alcohol when it is not synthesized directly.
  • the alcohol is useful for being functionalized, prior to incorporation of the PEG-lipid into a LNP, so that a binding moiety can be conjugated to it as a targeting moiety for the LNP (making it a tLNP).
  • the terminus of the PEG moiety and similar constructions, refers to the end of the PEG moiety that is not attached to the lipid.
  • a PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity, i.e. reducing the heterogeneity of a dispersion of LNPs. Additionally, a PEG moiety can impede binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the reticuloendothelial system.
  • the PEG-moiety can also be functionalized to serve as an attachment point for a targeting moiety. Conjugating a cell- or tissue-specific binding moiety to the PEG-moiety enables a tLNP to avoid the liver and bind to its target tissue or cell type, greatly increasing the proportion of LNP that reaches the targeted tissue or cell type. PEG- lipid can thus serve as means for inhibiting LNP binding, and PEG-lipid conjugated to a binding moiety can serve as means for LNP-targeting. [00269] As used herein, the term “functionalized PEG-lipid” and similar constructions refer generally to both the unreacted and reacted entities.
  • lipid composition of a LNP can be described referencing the reactive species even after conjugation has taken place (forming a tLNP).
  • a lipid composition can be described as comprising DSPE-PEG- maleimide and can be said to further comprise a binding moiety without explicitly noting that upon reaction to form the conjugate the maleimide will have been converted to a succinimide (or hydrolyzed succinimide).
  • the reactive group is bromomaleimide, after conjugation it will be maleimide.
  • Certain embodiments comprise a functionalized DSPE-PEG, for example, functionalized DSPE- PEG-2000. Certain embodiments comprise both DSG-PEG-2000 and functionalized DSPE- PEG-2000. In some instances, the functionalized PEG-lipid is functionalized with a maleimide moiety, for example, DSPE-PEG-2000-MAL. Analogously, in certain embodiments, DSG-PEG serves as the functionalized or functionalized and non- functionalized PEG-lipid.
  • the PEG-lipid and/or functionalized PEG-lipid comprises a scaffold selected from Formula S1, Formula S2, Formula S3, or Formula S4: Formula S1 Formula S2 Formula S3 Formula S4 wherein represents the points of ester connection with a fatty acid, and represents the point of ester (S1) or ether (S2, S3, and S4) formation with the PEG moiety.
  • the fatty acid esters are C 14 -C 20 straight-chain alkyl fatty acids.
  • the PEG moiety is functionalized and the fatty acid esters are C 16 -C 20 straight- chain alkyl fatty acids.
  • the straight-chain alkyl fatty acid is C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 .
  • the fatty acid esters are C 14 -C 20 symmetric branched-chain alkyl fatty acids.
  • the branched-chain alkyl fatty acid is C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 .
  • symmetric it is meant that each alkyl branch has the same number of carbons.
  • the branch is at the 3, 4, 5, 6, or 7 position of the fatty acid ester.
  • PEG-lipids built on scaffolds S1-S4 are disclosed in WO2023/196445A1 which is incorporated by reference for all that it teaches about PEG-lipids and their use.
  • Some embodiments of the disclosed ionizable cationic lipids have head groups with small ( ⁇ 250 Da) PEG moieties. These lipids are not what is meant by the term PEG- lipid as used herein. These small PEG moieties are generally too small to impede binding to a similar extent as the larger PEG moieties of the PEG-lipids disclosed above, though they will impact the lipophilicity of ionizable cationic lipid.
  • a functionalized PEG-lipid of a LNP or tLNP of this disclosure comprises one or more fatty acid tails, each of which is no shorter than C 16 nor longer than C 20 for straight-chain fatty acids. For branched chain fatty acids, tails no shorter than C 14 fatty acids nor longer than C 20 are acceptable. In some embodiments, fatty acid tails are C 16 . In some embodiments, the fatty acid tails are C 18 . In some embodiments, the functionalized PEG-lipid comprises a dipalmitoyl lipid.
  • the functionalized PEG-lipid comprises a distearoyl lipid.
  • the fatty acid tails serve as means to anchor the PEG-lipid in the tLNP to reduce or eliminate shedding of the PEG-lipid from the tLNP. This is a useful property for the PEG-lipid whether or not it is functionalized but has greater significance for the functionalized PEG-lipid as it will have a targeting moiety attached to it and the targeting function could be impaired if the PEG-lipid (with the conjugated binding moiety, such as an antibody) were shed from the tLNP.
  • an LNP or tLNP comprises about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol% PEG-lipid comprising functionalized and non-functionalized PEG-lipid.
  • an LNP or tLNP comprises DSG-PEG.
  • an LNP or tLNP comprises DMG-PEG or DPG-PEG.
  • an LNP or tLNP comprises DSPE-PEG.
  • the functionalized and non- functionalized PEG-lipids are not the same PEG-lipid, for example, the non-functionalized PEG-lipid can be a diacylglycerol and the functionalized PEG-lipid a diacyl phospholipid.
  • tLNP with such mixtures have reduced expression in the liver, possibly due to reduced uptake.
  • the functionalized PEG-lipid is DSPE-PEG and the non- functionalized PEG-lipid is DSG-PEG.
  • an LNP or tLNP comprises about 0.4 mol% to about 2.9 mol% or about 0.9 mol% to about 1.4 mol% non-functionalized PEG lipid.
  • an LNP or tLNP comprises about 1.4 mol% or 1.4 mol% non-functionalized PEG lipid. In some embodiments, an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol% or 0.1 mol% to 0.3 mol% functionalized lipid. In some instances, the functionalized lipid is DSPE-PEG. In certain instances, an LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% DSPE-PEG. In certain instances, an LNP or tLNP comprises 0.1 mol%, 0.2 mol%, or 0.3 mol% DSPE-PEG.
  • the functionalized PEG-lipid is conjugated to a binding moiety.
  • the phrase “is conjugated to” and similar constructions are meant to convey a state of being, that is, a structure, and not a process, unless context dictates otherwise.
  • Conjugation Any suitable chemistry can be used to conjugate the binding moiety to the PEG of the PEG-lipid, including maleimide (see Parhiz et al., J. Controlled Release 291:106, 2018) and click (see Kolb et al., Angewandte Chemie International Edition 40(11):2004, 2001; and Evans, Australian J. Chem.60(6):384, 2007) chemistries.
  • Reagents for such reactions include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid, PEG- dibenzocyclooctyne (DBCO), and lipid-PEG-azide. Further conjugations reactions make use of lipid-PEG-bromo maleimide, lipid-PEG-alkylnoic amide, PEG-alkynoic imide, and lipid- PEG-alkyne reactions, as disclosed in U.S.
  • the binding moiety e.g., an antibody
  • SATA N-succinimidyl S-acetylthioacetate
  • SATA is then deprotected, for example, using 0.5 M hydroxylamine followed by removal of the unreacted components by G-25 Sephadex Quick Spin Protein columns (Roche Applied Science, Indianapolis, IN).
  • the reactive sulfhydryl group on the binding moiety is then conjugated to maleimide moieties on LNPs of the disclosure using thioether conjugation chemistry.
  • an alkyne can be added to a sulfhydryl or an epsilon amino of a lysine to participate in a click chemistry reaction.
  • Purification can be performed using Sepharose CL-4B gel filtration columns (Sigma-Aldrich).
  • tLNPs LNPs conjugated with a targeting antibody
  • Others have conjugated antibody to free functionalized PEG-lipid and then incorporated the conjugated lipid into pre-formed LNP. However, it was found that this procedure is more controllable and produces more consistent results.
  • C-terminal extensions of native or artificial sequences containing a particularly accessible cysteine residue are commonly used. Partial reduction of cysteine bonds in an antibody, for example, with tris(2- carboxy)phosphine (TCEP), can also generate thiol groups for conjugation which can be site-specific under defined conditions with an amenable antibody fragment.
  • TCEP tris(2- carboxy)phosphine
  • the C-terminal extension can contain a sortase A substrate sequence, LPXTG (SEQ ID NO: 1) which can then be functionalized in a reaction catalyzed by sortase A and conjugated to the PEG-lipid, including through click chemistry reactions (see, for example, Moliner-Morro et al., Biomolecules 10(12):1661, 2020 which is incorporated by reference herein for all that it teaches about antibody conjugations mediated by the sortase A reaction and/or click chemistry).
  • LPXTG SEQ ID NO: 1
  • AJICAP reagents are modified affinity peptides that bind to specific loci on the Fc and react with an adjacent lysine residue to form an affinity peptide conjugate of the antibody. The peptide is then cleaved with base to leave behind a thiol-functionalized lysine residue which can then undergo conjugation through maleimide or haloamide reactions, for example).
  • the binding moiety is conjugated to the PEG moiety of the PEG-lipid through a thiol modified lysine residue.
  • the conjugation is through a cysteine residue in a native or added antibody sequence.
  • the tLNP of the various disclosed aspects comprise a binding moiety, such as an antibody or antigen binding domain thereof or a cell surface receptor ligand.
  • a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide or peptide, carbohydrate, nucleic acid, or combinations thereof capable of specifically binding to a target or multiple targets.
  • a binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest.
  • Exemplary binding moieties of this disclosure include an antibody, a Fab ⁇ , F(ab ⁇ ) 2 , Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), a single domain antibody, VHH, VNAR, sdAb, nanobody, receptor ectodomain or ligand-binding portions thereof, or ligand (e.g., cytokines, chemokines).
  • Fab antigen binding fragment
  • a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand.
  • a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders.
  • assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, biolayer interferometry, and surface plasmon resonance.
  • a binding moiety such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains.
  • scFv immunoglobulin light and heavy chain variable domains
  • a binding moiety can be an antibody or an antigen-binding portion thereof; an antigen; a ligand-binding domain of a receptor; or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. [00282] In some embodiments, a binding moiety comprises an antibody or an antigen- binding portion thereof.
  • antibody refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen, termed complementarity determining regions (CDRs).
  • antibody can thus refer to intact or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (from camels, alpacas, llamas, etc.) produce antibodies with both the canonical structure and antibodies comprising only heavy chains.
  • the variable region of the camelid heavy chain-only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody.
  • Antigen binding fragments and constructs of antibodies include F(ab) 2 , F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH.
  • Such elements can be combined to produce bi- and multi-specific reagents, including various immune cell engagers, such as BiTEs (bi-specific T-cell engagers).
  • various immune cell engagers such as BiTEs (bi-specific T-cell engagers).
  • Antibodies can be obtained through immunization, selection from a na ⁇ ve or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that can be used as binding moieties are known in the art.
  • WO2024040195A1 is also a source of sequence and other information for a wide range of antibodies with specificity for various cell surface antigens of immune system cells and cancer cells and is incorporated herein by reference for all that it teaches about individual antibodies and the antigens they bind.
  • An antibody or other binding moiety “specifically binds” a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/Molar or 1/M) equal to or greater than 10 5 M -1 , while not significantly binding other components present in a test sample.
  • Ka i.e., an equilibrium association constant of a particular binding interaction with units of 1/Molar or 1/M
  • Binding domains can be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof).
  • “High affinity” binding domains refer to those binding domains with a Ka of at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 , preferably at least 10 8 M -1 or at least 10 9 M -1 .
  • “Low affinity” binding domains refer to those binding domains with a Ka of up to 10 8 M -1 , up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 .
  • affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 -5 M to 10 -13 M).
  • Kd equilibrium dissociation constant
  • Affinities of binding domain polypeptides and fusion proteins according to this disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., 1949, Ann. N.Y. Acad. Sci.51:660; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent).
  • a diabody is a type of scFv dimer in which each chain consists of the V H and V L regions connected by a small peptide linker that is too short to allow pairing between the two domains of the same chain.
  • a BiTE is a fusion protein having two scFvs of different antibodies, usually an antibody for a tumor-associated antigen and antibody for CD3, on a single peptide chain, thus forming a cytolytic synapse between T cells and target antigen-bearing cells.
  • the term "antigen-binding portion" can refer to a portion of an antibody as described that possesses the ability to specifically recognize, associate, unite, or combine with a target molecule.
  • An antigen-binding portion includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a specific antigen.
  • antibodies and antigen-binding portions thereof constitute means for binding to the surface molecule on a cell.
  • the cell can be an immune cell, a leukocyte, a lymphocyte, a monocyte, a stem cell, an HSC or an MSC, according to the specificity of the antibody.
  • the antibody or antigen-binding portion thereof can be derived from a mammalian species, for example, mice, rats, or human.
  • Antibody variable regions can be those arising from one species, or they can be chimeric, containing segments of multiple species possibly further altered to optimize characteristics such as binding affinity or low immunogenicity. For human applications, it is desirable that the antibody has a human sequence.
  • the antibody or antigen-binding portion thereof can be humanized to reduce immunogenicity in a human subject.
  • the non- human antibody can be humanized, e.g., through CDR grafting, in which the CDRs from the non-human antibody are placed into the respective positions in a framework of a compatible human antibody.
  • an antibody in which only the constant region of the non- human antibody is replaced with human sequence are commonly referred to as chimeric antibodies in distinction to humanized antibodies.
  • the antibody or antigen-binding portion thereof is non- immunogenic.
  • the antibody can be modified in its Fc region to reduce or eliminate secondary functions, such as FcR engagement, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement- dependent cytotoxicity (CDC); this is often referred to as an Fc silenced antibody.
  • a binding moiety density on the LNP (or tLNP) can be defined according to the ratio of antibody (binder) to mRNA (w/w) either based on the amount of antibody input in the conjugation reaction or as measured in the LNP or (tLNP) after conjugation.
  • preferred ratios are about 0.3 to about 1.0, about 0.3 to about 0.7, about 0.3 to about 0.5, about 0.5 to about 1.0, and about 0.5 to about 0.7 for either the input or final measured binder ratio.
  • a LNP or tLNP has an antibody ratio of 0.3 to 1.0, 0.3 to 0.7, 0.3 to 0.5, 0.5 to 1.0, and 0.5 to 0.7 for either the input or final measured binder ratio.
  • a LNP or tlNP comprise a binding moiety that is a F(ab’) or F(ab’) analog.
  • F(ab’) and F(ab’)-like formats offer certain advantages as targeting moieties for a tLNP.
  • F(ab’) analog has been adopted herein to refer to engineered sequences comprising amino acid substitutions and/or that have been truncated and to distinguish them from the paradigmatic natural sequence.
  • F(ab’) are smaller than whole antibodies which can be advantageous in manufacturing.
  • targeting moiety on a tLNP their antigen binding domain is further from the LNP surface than, for example, a scFv, which can facilitate interaction with the target cell surface.
  • F(ab’) molecules have cysteine residues in the partial hinge region that can be readily conjugated to a functionalized PEG-lipid (for example, a maleimide- functionalized PEG-lipid).
  • a functionalized PEG-lipid for example, a maleimide- functionalized PEG-lipid
  • the F(ab’) can be engineered so that there is unique accessible cysteine enabling for site-specific conjugation which is desirable for product consistency. This can be accomplished with recombinant DNA technology by truncating the hinge region of the F(ab’) or by changing cysteine residues to another amino acid, such as serine, or both.
  • the hinge region cysteines can form a cystine with another F(ab’) molecule forming an F(ab’) 2 which would make the cysteine unavailable for conjugation to an LNP (more specifically, a functionalized lipid thereof). This can be prevented by processing the F(ab’) under mildly reducing conditions, however, this poses a risk of disrupting the interchain disulfide bond between CL and CH1. That risk can be obviated by relocating the interchain bond to a less accessible region in the molecule.
  • Some aspects combine constant regions of an F(ab’) or F(ab’) analog with a humanized immunoglobulin antigen binding domain derived from the anti-CD8 ⁇ antibody CT8 as disclosed herein.
  • Humanized anti-CD8 antigen binding domains and antibodies and F(ab’) analog design useful as LNP targeting moieties are disclosed, for example, in International Patent Application No. PCT/US2024/060426, filed December 16, 2024, which is incorporated for reference for all that it teaches about anti-CD8 antigen binding domains and antibodies and F(ab’) analog designs.
  • Some aspects combine constant regions of an F(ab’) analog with the antigen binding domain of an anti-CD8 antibody.
  • the anti-CD8 antigen binding domain recognizes the CT8 epitope.
  • the anti-CD8 antigen binding domain is derived from YTC182.20, TRX2, or CT8.
  • the anti-CD8 antigen binding domain comprises a humanized immunoglobulin antigen binding domain derived from the anti-CD8 ⁇ antibody CT8 as disclosed herein.
  • an F(ab’) analog engineered as disclosed herein is conjugated to an LNP but is generic with respect to the variable domains of the F(ab’) analog and its specificity.
  • an F(ab’) or F(ab’) analog constant regions are combined with the antigen binding domain of an anti-CD8 antibody which is conjugated to an LNP.
  • the anti-CD8 antigen binding domain recognizes the CT8 epitope.
  • the anti-CD8 antigen binding domain is derived from YTC182.20, TRX2, or CT8.
  • the anti-CD8 antigen binding domain comprises a humanized immunoglobulin antigen binding domain derived from the anti-CD8 ⁇ antibody CT8 as disclosed herein.
  • the F(ab’) analog comprises a relocated interchain disulfide bond, for example, a C ⁇ S162C substitution paired with an IgG1 or IgG4 CH1 F174C substitution.
  • a LNP or tLNP comprises a binding moiety derived from an anti-CD40* antibody, an anti-LRRC15 ⁇ antibody, an anti-CTSK antibody, an anti- ADAM12 antibody, an anti-ITGA11 antibody, an anti-FAP* ⁇ antibody, an anti-NOX4 antibody, an anti-SGCD antibody, an anti-SYNDIG1 antibody, an anti-CDH11 antibody, an anti-PLPP4 antibody, an anti-SLC24A2 antibody, an anti-PDGFRB* antibody, an anti-THY1 antibody, an anti-ANTXR1 antibody, an anti-GAS1 antibody, an anti-CALHM5 antibody, an anti-SDC1* antibody, an anti-HER2* ⁇ antibody, an anti-
  • a LNP comprises a binding moiety specific for an immune cell antigen selected from CD1, CD2* ⁇ , CD3* ⁇ , CD4* ⁇ , CD5 ⁇ , CD7 ⁇ , CD8 ⁇ , CD11b, CD14 ⁇ , CD16, CD25 ⁇ , CD26*, CD119, CD126, CD137 (41BB) ⁇ , CD150, CD153, CD161, CD166, CD183 (CXCR3), CD183 (CXCR5), CD223 (LAG-3)* ⁇ , CD254, CD275, CD45RA, CTLA-4* ⁇ * ⁇ , DEC205, OX40 ⁇ , PD-1* ⁇ , GITR ⁇ , TIM-3* ⁇ , , FasL*, IL18R1, ICOS (CD278), leu-12, TCR ⁇ , TLR1, TLR2 ⁇ , TLR3*, TLR4
  • a tLNP comprises a binding moiety specific for an HSC surface molecule selected from CD117 ⁇ , CD34*, CD44*, CD90 (Thy1) , CD105, CD133, BMPR2, and Sca-1; or specific for an MSC surface molecules selected from CD70*, CD105, CD73, Stro-1, SSEA-3, SSEA-4, CD271, CD146, GD2* ⁇ , SUSD2, Stro-4, MSCA-1, CD56, CD200* ⁇ , PODXL, CD13, CD29*, CD44*, and CD10.
  • a binding moiety is an antibody or antigen-binding portion thereof.
  • WO2024040195A1 filed August 17, 2023, each of which is incorporated herein by reference for all that it teaches about individual antibodies and the antigens they bind.
  • the following paragraphs provide non-exhaustive examples of known antibodies that bind to cell surface markers/antigens on immune cells (lymphocytes and monocytes) and stem cells (HSC and MSC). These antibodies or the antigen binding domains thereof can be used as binding moieties to target the disclosed LNP. Similarly, these antibodies can contribute their antigen binding domains to immune cell reprogramming agents such as CARs and ICEs.
  • an immune cell reprogramming agent While typically an immune cell reprogramming agent is expressed in an immune cell, one call also express a biological response modifier (conditioning agent) or an immune cell reprogramming agent, such as an ICE, in a tumor cell.
  • a biological response modifier conditioning agent
  • an immune cell reprogramming agent such as an ICE
  • the immune and stem cell surface markers that can serve as a targeted antigen of a tLNP can also usefully be a target of an immune cell reprogramming agent when the cell expressing that antigen has a role in the pathology of some disease or condition.
  • Collectively these antibodies and polypeptides comprising the antigen binding domains thereof constitute means for binding cell surface markers or means for binding immune and stem cells.
  • CD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD2 antibody.
  • CD2 contains three well-characterized epitopes (T11.1, T11.2, and T11.3/CD2R).
  • T11.3/CD2R are membrane proximal and exposure is increased upon T cell activation and CD2 clustering.
  • the anti-CD2 antigen binding domain is derived from, RPA-2.10; OKT11, UMCD2, 0.1, and 3T4-8B5 (T11.1 epitope); 9.6 and 1OLD2-4C1 (T11.2 epitope); 1Mono2A6 (T11.3 epitope), siplizumab (T11.2/T11.3 epitope), HuMCD2, TS2/18, TS1/8, AB75, LT-2, T6.3, MEM-65, OTI4E4, or an antigen-binding portion thereof.
  • CD2 can be used as a CD2 binding moiety as can alefacept, a CD58-Fc fusion.
  • CD3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD3 antibody.
  • the antigen binding domain is derived from muromonab-CD3 (OKT3), teplizumab, otelixizumab, visilizumab, cevostamab, teclistamab, elranatamab pavurutamab, vibecotamab, odronextamab, or an antigen-binding portion thereof.
  • CD4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD4 antibody.
  • the antigen binding domain is derived from ibalizumab, inezetamab, semzuvolimab, zanolimumab, tregalizumab, UB-421, priliximab, MTRX1011A, cedelizumab, clenoliximab, keliximab, M-T413, TRX1, hB-F5, MAX.16H5, IT208, or an antigen-binding portion thereof.
  • CD5 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD5 antibody.
  • the antigen binding domain is derived from 5D7, UCHT2, L17F12, H65, HE3, OKT1, MAT304, as well as those disclosed in WO1989006968, WO2008121160, US8,679,500, WO2010022737, WO2019108863, WO2022040608, or WO2022127844, each of which is incorporated by reference for all that they teach about anti-CD5 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD5.
  • CD7 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD7 antibody.
  • the antigen binding domain is derived from TH-69, 3A1E, 3A1F, Huly- m2, WT1, YTH3.2.6, T3-3A1, grisnilimab, as well as those disclosed in US10,106,609, WO2017213979, WO2018098306, US11447548, WO2022136888, WO2020212710, WO2021160267, WO2022095802, WO2022095803, WO2022151851, or WO2022257835 each of which is incorporated by reference for all that they teach about anti-CD7 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD7.
  • CD8 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD8 antibody.
  • the antigen binding domain is derived from crefmirlimab (IAB22M), 3B5, SP-16, LT8, 17D8, MEM-31, MEM-87, RIV11, UCHT4, YTC182.20, RPA-T8, OKT8, SK1, 51.1, TRX2, MT807-R1, HIT8 ⁇ , C8/144B, RAVB3, SIDI8BEE, BU88, EPR26538-16, 2ST8.5H7, as well as those disclosed in US10,414,820, WO2015184203, WO2017134306, WO2019032661, WO2020060924, US10,730,944, WO2019033043, WO2021046159, WO2021127088, WO2022081516, US11,535,869, or
  • humanized anti-CD8 antibodies are described in U.S. Provisional Patent Application Number 63/610,917, filed on December 15, 2023, U.S. Provisional Patent Application Number 63/654,930, filed on May 31, 2024, U.S. Provisional Patent Application Number 63/708,461, filed on October 17, 2024, and International Patent Application Number PCT/US2024/060426, filed on December 16, 2024, each of which is incorporated by reference for all that it teaches about these humanized anti-CD8 antibodies and their properties, or an antigen-binding portion thereof.
  • Each of the foregoing anti-CD8 antibodies constitutes a means for binding CD8.
  • a tLNP is targeted to CD10 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD10 antibody.
  • the antigen binding domain is derived from the one produced by the hybridoma represented by Accession No. NITE BP-02489 (disclosed in WO2018235247 which is incorporated by reference for all that they teach about anti-CD10 antibodies and their properties), FR4D11, or REA877, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD10.
  • CD11b is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD11b antibody.
  • the antigen binding domain is derived from ASD141 or MAB107 as well as those disclosed in US20150337039, US10,738,121, WO2016197974, US10,919,967, or WO2022147338 each of which is incorporated by reference for all that they teach about anti-CD11b antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD11b.
  • CD13 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD13 antibody.
  • CD13 is also known as aminopeptidase N (APN).
  • the antigen binding domain is derived from MT95-4 or Nbl57 (disclosed in WO2021072312 which is incorporated by reference for all that they teach about anti-CD13 antibodies and their properties), as well as those disclosed in WO2023037015 which is incorporated by reference for all that it teaches about anti-CD13 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD13.
  • CD14 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD14 antibody.
  • the antigen binding domain is derived from atibuclimab or r18D11 as well as those disclosed in WO2018191786 or WO2015140591 each of which is incorporated by reference for all that they teach about anti-CD14 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD14.
  • CD16a is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD16a antibody.
  • the antigen binding domain is derived from AFM13, sdA1, sdA2, or hu3G8-5.1-N297Q as well as those disclosed in US11535672, WO2018158349, WO2007009065, US10385137, WO2017064221, US10,758,625, WO2018039626, WO2018152516, WO2021076564, WO2022161314, or WO2023274183 each of which is incorporated by reference for all that they teach about anti-CD16A antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD16a.
  • CD25 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD25 antibody.
  • the antigen binding domain is derived from daclizumab, basiliximab, camidanlumab, tesirine, inolimomab, RO7296682, HuMax-TAC, CYT-91000, STI-003, RTX-003, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD25.
  • CD28 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD28 antibody.
  • the antigen binding domain is derived from GN1412, acazicolcept, lulizumab, prezalumab, theralizumab, FR104CD, and davoceticept, as well as those disclosed in US8,454,959, US8,785,604, US11,548,947, US11,530,268, US11,453,721, US11,591,401, WO2002030459, WO2002047721, US20170335016, US20200181260, US11608376, WO2020127618, WO2021155071, or WO2022056199 each of which is incorporated by reference for all that they teach about anti-CD28 antibodies and their properties, or an antigen-binding portion thereof.
  • CD29 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD29 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from OS2966, 6D276, 12G10, REA1060, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD29.
  • CD32A is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD32A antibody.
  • the antigen binding domain is derived from VIB9600, humanized IV.3, humanized AT-10, or MDE-8 as well as those disclosed in US9,688,755, US9,284,375, US9,382,321, US11306145, or WO2022067394 each of which is incorporated by reference for all that they teach about anti-CD32A antibodies and their properties, or an antigen- binding portion thereof. Each of these constitutes a means for binding CD32A.
  • CD34 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD34 antibody.
  • the antigen binding domain is derived from h4C8, 9C5, 2E10, 5B12, REA1164, C5B12, C2e10, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD34.
  • CD40 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD40 antibody.
  • the antigen binding domain is derived from cifurtilimab, sotigalimab, iscalimab, dacetuzumab, selicrelumab, bleselumab, lucatumumab, or mitazalimab as well as those disclosed in US10633444, each of which is incorporated by reference for all that they teach about anti-CD40 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD40.
  • CD44 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD44 antibody.
  • the antigen binding domain is derived from RO5429083, VB6-008, PF-03475952, or RG7356, as well as those disclosed in WO2008144890, US8,383,117, WO2008079246, US20100040540, WO2015076425, US9,220,772, US20140308301, WO2020159754, WO2021160269, WO2021178896, WO2022022749, WO2022022720, or WO2022243838, each of which is incorporated by reference for all that they teach about anti-CD44 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD44.
  • CD45 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD45 antibody.
  • the antigen binding domain is derived from apamistamab, BC8- B10, as well as those disclosed in WO2023183927, WO2023235772, US7,825,222, WO2017009473, WO2021186056, US9,701,756, US9,701,756, WO2020092654, WO2022040088, WO2022040577, WO2022064191, WO2022063853, or WO2024064771, each of which is incorporated by reference for all that they teach about anti-CD45 antibodies and their properties, or an antigen-binding portion thereof.
  • CD56 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD56 antibody.
  • the antigen binding domain is derived from lorvotuzumab, adcitmer, or promiximab, as well as those disclosed in WO2012138537, US10,548,987, US10,730,941, or US20230144142, each of which is incorporated by reference for all that they teach about anti-CD56 antibodies and their properties, or an antigen-binding portion thereof.
  • Each of these constitutes a means for binding CD56.
  • CD64 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD64 antibody.
  • the antigen binding domain is derived from HuMAb 611 or H22 as well as those disclosed in US7,378,504, WO2014083379, US20170166638, or WO2022155608 each of which is incorporated by reference for all that they teach about anti- CD64 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD64.
  • CD68 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD68 antibody.
  • the antigen binding domain is derived from Ki-M7, PG-M1, 514H12, ABM53F5, 3F7C6, 3F7D3, Y1/82A, EPR20545, CDLA68-1, LAMP4-824, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD68.
  • CD70 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD70 antibody.
  • the antigen binding domain is derived from cusatuzumab, vorsetuzumab, MDX-1203, MDX-1411, AMG-172, SGN-CD70A, ARX305, PRO1160, as well as those disclosed in US9,765,148, US8,124,738, IS10,266,604, WO2021138264, US9,701,752, US10,108,123, WO2014158821, US10,689,456, WO2017062271, US11,046,775, US11,377,500, WO2021055437, WO2021245603, WO2022002019, WO2022078344, WO2022105914, WO2022143951, WO2023278520, WO2022226317, WO2022262101, US11,613,584, or WO2023072307, each of which is incorporated by reference for all that they teach about anti-CD70 antibodies and their properties, or an antigen-binding portion
  • CD73 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD73 antibody.
  • the antigen binding domain is derived from oleclumab, uliledlimab, mupadolimab, AK119, IBI325, BMS-986179, NZV930, JAB-BX102, Sym024, TB19, TB38, HBM1007, 3F7, mAb19, Hu001-MMAE, IPH5301, or INCA00186, as well as those disclosed in US9,938,356, US10,584,169, WO2022083723, WO2022037531, WO2021213466, WO2022083049, US10,822,426, WO2021259199, US10,100,129, US11,312,783, US11,174,319, US11,634,500, WO
  • CD90 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD90 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REA897, OX7, 5E10, K117, L127, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD90.
  • CD105 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD105 antibody.
  • the antigen binding domain is derived from carotuximab, TRC205, or huRH105, as well as those disclosed in US8,221,753, US9,926,375, WO2010039873, WO2010032059, WO2012149412, WO2015118031, WO2021118955, US20220233591, or US20230075244, each of which is incorporated by reference for all that they teach about anti-CD105 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD105.
  • CD117 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD117 antibody.
  • the antigen binding domain is derived from briquilimab, barzolvolimab, CDX-0158, LOP628, MGTA-117, NN2101, CK6, JSP191, Ab85, 104D2, or SR1, as well as those disclosed in US7,915,391, WO2022159737, US9540443, WO2015050959, US9,789,203, US8,552,157, US10,406,179, US9,932,410, WO2019084067, WO2020219770, US10,611,838, WO2020076105, WO2021107566, US11,208,482, WO2021044008, WO2021099418, WO2022187050, or WO2023026791, WO2021188590, each of which is incorporated by reference for all that they teach about anti-CD117 antibodies and their properties, or an antigen-binding portion thereof.
  • CD133 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD133 antibody.
  • the antigen binding domain is derived from AC133, 293C3, CMab- 43, or RW03, as well as those disclosed in WO2018045880, US8,722,858, US9,249,225, WO2014128185, US10,711,068, US10,106,623, WO2018072025, or WO2022022718, each of which is incorporated by reference for all that they teach about anti-CD133 antibodies and their properties, or an antigen-binding portion thereof.
  • CD137 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD137 antibody.
  • CD137 is also known as 4-1BB.
  • the antigen binding domain is derived from YH004, urelumab (BMS-663513), utomilumab (PF-05082566), ADG106, LVGN6051, PRS-343, as well as those disclosed in WO2005035584, WO2012032433, WO2017123650, US11,203,643, US11,242,395, US11,555,077, US20230067770, US11,535,678, US11,440,966, WO2019092451, US10,174,122, US11,242,385, US10,716,851, WO2020011966, WO2020011964, or US11,447,558, each of which is incorporated by reference
  • CD146 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD146 antibody.
  • the antigen binding domain is derived from imaprelimab, ABX- MA1, huAA98, M2H, or IM1-24-3, as well as those disclosed in US10,407,506, US10,414,825, US6,924,360, US9,447,190, WO2014000338, US9,782,500, WO2018220467, US11,427,648, WO2019133639, WO2019137309, WO2020132190, or WO2022082073, each of which is incorporated by reference for all that they teach about CD146 antibodies and their properties, or an antigen-binding portion thereof.
  • CD166 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD166 antibody.
  • the antigen binding domain is derived from praluzatamab, AZN- L50, REA442, or AT002, as well as those disclosed in US10,745,481, US11,220,544, or WO2008117049, each of which is incorporated by reference for all that they teach about CD166 antibodies and their properties, or an antigen-binding portion thereof.
  • Each of these constitutes a means for binding CD166.
  • CD200 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD200 antibody.
  • the antigen binding domain is derived from samalizumab, OX-104, REA1067, B7V3V2, HPAB-0260-YJ, or TTI-CD200, as well as those disclosed in WO2007084321 or WO2019126536, each of which is incorporated by reference for all that they teach about CD200 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD200.
  • CD205 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD205 antibody.
  • CD205 is also known as DEC205.
  • the antibody comprises 3G9- 2D2 (a component of CDX-1401) or LY75_A1 (a component of MEN1309) as well as those disclosed in US8,236,318, US10,081,682, or US11,365,258, each of which is incorporated by reference for all that they teach about anti-CD205 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD205.
  • CD271 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD271 antibody.
  • the antigen binding domain is derived from REA844 or REAL709 as well as those disclosed in WO2022166802 which is incorporated by reference for all that it teaches about anti-CD271 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD271.
  • BMPR2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-BMPR2 antibody.
  • the antigen binding domain is derived from TAB-071CL (Creative Biolabs catalog no.) as well as those disclosed in US11,292,846 or WO2021174198, each of which is incorporated by reference for all that they teach about anti-BMPR2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding BMPR2.
  • claudin 18.2 (CLDN 18.2) is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-claudin 18.2 antibody.
  • the antigen binding domain is derived from zolbetuximab, osemitamab, RC118, IBI-343, AZD0901, M108, SYSA1801, TORL-2- 307-ADC, LM-302, ASKB589, gresonitamab, SPX-101, SKB315, Q-1802, GIVASTOMIG, LCAR-C18S, SOT102, CT041 as well as those disclosed in WO2013167259, WO2021032157, WO2021254481, WO2022007808, WO2021008463, WO2022111616, WO2018006882, WO2020147321, WO2019219089, US20200040101, WO2020025792, WO2020139956, WO2020135201, US20240228610, WO2021218874, WO2021027850, WO2021129765, WO2022068854, WO2021111003, each
  • CTLA-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CTLA-4 antibody.
  • the antigen binding domain is derived from botensilimab, ipilimumab, nurulimab, quavonlimab, tremelimumab, zalifrelimab, ADG116, ADG126, ADU- 1604, AGEN1181, BCD-145, BMS-986218, BMS-986249, BT-007, CS1002, GIGA-564, HBM4003, IBI310 JK08, JMW-3B3, JS007, KD6001, KN044, ONC-392, REGN4659, TG6050, XTX101, YH001, or an antigen-binding portion thereof.
  • GD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GD2 antibody.
  • the antigen binding domain is derived from dinutuximab, ganglidiximab, naxitamab, nivatrotamab, EMD 273063, hu14.18k322A, MORAb-028, 3F8BiAb, BCD-245, KM666, ATL301, Ektomab, as well as those disclosed in US9,777,068, US9,315,585, WO2004055056, US9,617,349, US9,493,740, US20210002384, US8507657, WO2001023573, WO2012071216, WO2018010846, US8,951,524, WO2023280880, US9,856,324, WO2015132604,
  • GITR is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GITR antibody.
  • the antigen binding domain is derived from ragifilimab, TRX518, MK- 4166, AMG 228, MEDI1873, BMS-986156, REGN6569, ASP1951, MK-1248, FRA154, GWN323, JNJ-64164711, ATOR-1144, or an antigen-binding portion thereof.
  • the antigen binding domain is derived from ragifilimab, TRX518, MK- 4166, AMG 228, MEDI1873, BMS-986156, REGN6569, ASP1951, MK-1248, FRA154, GWN323, JNJ-64164711, ATOR-1144, or an antigen-binding portion thereof.
  • Each of these constitutes a means for binding GITR.
  • a low affinity IL-2 receptor is a targeted cell surface antigen (CD122 and/or CD132) and a binding moiety comprises the antigen binding domain of an anti-IL-2 receptor antibody.
  • the antiCD122 antibody comprises ANV419, FB102, MiK-Beta-1 and the anti CD122 antibodies disclosed in WO2011127324, WO2017021540, WO2022212848, WO2022221409, WO2023078113, US20230272090, WO2024073723, or an antigen-binding portion thereof.
  • the anti-CD132 antibody comprises REGN7257 and the anti- CD132 antibodies disclosed in WO2020160242, WO2017021540, WO2022212848, WO2023078113, US20230272089, or an antigen-binding portion thereof.
  • Each of these constitutes a means for binding the low affinity IL-2 receptor (CD122 or CD132, as appropriate)
  • a high affinity IL-2 receptor is a targeted cell surface antigen (CD25) and a binding moiety comprises the antigen binding domain of an anti-IL-2 receptor antibody.
  • the antigen binding domain is derived from daclizumab, basiliximab, camidanlumab, vopitug, inolimomab, HuMAx-TAC, Xenopax, STI-003, RA8, RTX-003, and the anti-CD25 antibodies disclosed in WO2023031403, WO2006108670, WO2019175223, WO2019175215, WO2019175226, WO2004045512, WO2022104009, WO2020102591, or an antigen-binding portion thereof. Each of these constitutes a means for binding the high affinity IL-2 receptor (CD25).
  • IL-7 receptor is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-7 receptor antibody.
  • the antigen binding domain is derived from PF-06342674, GSK2618960, OSE-127, lusvertikimab, bempikibart, and the anti-CD127 antibodies disclosed in WO2011104687, WO2011094259, WO2013056984, WO2015189302, WO2017062748, WO2020154293, WO2020254827, WO2021222227, WO2023201316, or an antigen-binding portion thereof.
  • IL-12 receptor is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-12 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from CBYY- I0413, REA333, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-12 receptor.
  • IL-15 receptor ⁇ is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-15 receptor ⁇ antibody.
  • the antigen binding domain is derived from MAB1472-100, MAB5511, JM7A4, 5E3E1, JM7A4, 2639B, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-15 receptor ⁇ .
  • IL-18 receptor ⁇ is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-18 receptor ⁇ antibody.
  • the antigen binding domain is derived from H44, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-18 receptor ⁇ .
  • IL-21 receptor is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-21 receptor antibody.
  • the antigen binding domain is derived from 1D1C2, 19F5, 18A5, REA233, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-21 receptor ⁇ .
  • LAG-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-LAG-3 antibody.
  • the antigen binding domain is derived from relatlimab, tebotelimab, bootszelimab, fianlimab, miptenalimab, HLX26, ieramilimab, GSK2831781, INCAGN2385, RO7247669, encelimab, FS118, SHR-1802, Sym022, IBI110, IBI323, bavunalimab, EMB-02, ABL501, INCA32459, AK129, or an antigen-binding portion thereof. Each of these constitutes a means for binding LAG-3.
  • MSCA-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti- MSCA-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REAL219, W8B2, X9C3, or an antigen-binding portion thereof. Each of these constitutes a means for binding MSCA-1.
  • OX40 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-OX40 antibody.
  • the antigen binding domain is derived from MEDI6469, ivuxolimab, rocatinlimab, GSK3174998, BMS-986178, vonlerizumab, INCAGN1949, tavolimab, BGB-A445, INBRX-106, BAT6026, telazorlimab, ATOR-1015, MEDI6383, cudarolimab, FS120, HFB301001, EMB-09, HLX51, Hu222, ABM193, or an antigen-binding portion thereof.
  • Each of these constitutes a means for binding OX40.
  • PD-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-PD-1 antibody.
  • the antigen binding domain is derived from nivolumab, pembrolizumab, camrelizumab, torpalimab, sintilimab, tislelizumab, cemiplimab, spartalizumab, serplulimab, cadonilimab, penpulimab, dostarlimab, zimberelimab, retifanlimab, pucotenlimab, pidilizumab, pidilizumab, balstilimab, ezabenlimab, AK112, geptanolimab, cetrelimab, prolgolimab, tebotelimab, sasanlimab, SG001, vudalimab, MEDI
  • PODXL is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-PODXL antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MAI1738, HPAB- 3334LY, HPAB-MO612-YC, REA246, REA157, or an antigen-binding portion thereof. Each of these constitutes a means for binding PODXL.
  • Sca-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Sca-1 antibody.
  • the antigen binding domain is derived from CPP324-1-18, 2D4- C9-F1, AMM22070N, or an antigen-binding portion thereof. Each of these constitutes a means for binding SCA-1.
  • SSEA-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SSEA-3 antibody.
  • the antigen binding domain is derived from MC631, 2A9, 8A7, ND- 742, 3H420, as well as those disclosed in US11,643,456 or WO2021138378, each of which is incorporated by reference for all that they teach about anti-SSEA-3 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding SSEA-3.
  • SSEA-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SSEA-4 antibody.
  • the antigen binding domain is derived from ch28/11, REA101, MC-813-70, ND-942-80, as well as those disclosed in US11,446,379, US10,273,295, US11,643,456, WO2019190952, or WO2021044039, each of which is incorporated by reference for all that they teach about anti-SSEA-4 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding SSEA-4.
  • Stro-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Stro-1 antibody.
  • the antigen binding domain is derived from STRO-1, TUSP-2, as well as those disclosed in US20130122022, which is incorporated by reference for all that it teaches about anti-Stro-1 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding Stro-1.
  • Stro-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Stro-4 antibody.
  • the antigen binding domain is derived from STRO-4, efungumab, 4C5, as well as those disclosed in US7,722,869, US20110280881, US9,115,192, US10,273,294, US10,457,726, WO2023091148, each of which is incorporated by reference for all that they teach about anti-Stro-4 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding Stro-4 (also known as heat shock protein-90).
  • SUSD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SUSD2 antibody.
  • the antigen binding domain is derived from REA795, CBXS-3571, CBXS-1650, CBXS-1989, CBXS-1671, CBXS1990, CBXS-3676, 1279B, EPR8913(2), W5C5, or an antigen-binding portion thereof.
  • TIM-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-TIM-3 antibody.
  • the antigen binding domain is derived from TQB2618, sabatolimab, cobolimab, RO7121661, INCAGN02390, AZD7789, surzebiclimab, LY3321367, Sym023, BMS-986258, SHR-1702, LY3415244, LB1410, or an antigen-binding portion thereof. Each of these constitutes a means for binding TIM-3.
  • TREM2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-TREM2 antibody.
  • the antigen binding domain is derived from PI37012 as well as those disclosed in US10,508,148, US10,676,525, WO2017058866, US11,186,636, US11,124,567, WO2020055975, US11,492,402, WO2020121195, WO2023012802, WO2021101823, WO2023047100, WO2022032293, WO2022241082, WO2023039450, or WO2023039612, each of which is incorporated by reference for all that they teach about anti-TREM2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding TREM2.
  • G protein-coupled receptor, class C, group 5, member D is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GPRC5D antibody.
  • the antigen binding domain of an anti-GPRC5D antibody is derived from talquetamab, forimtamig, BMS-986393, IBI-3003, QLS32015, SIM0500, or EPR28376-41, or is disclosed in WO2018017786, WO2016090329, WO2022174813, WO2023236889, WO2018147245, WO2024079015, WO2019154890, WO2021018859, WO2021018925, WO2020092854, WO2024031091, WO2020148677, WO2022175255, WO2022222910, WO2022247804, WO2022247756, WO2023078382, WO20
  • FCRL5 (CD307E) is a targeted cell surface antigen and binding moiety comprises the antigen binding domain of an anti-FCRL5 antibody.
  • the antigen binding domain of an anti-FCRL5 antibody is derived from cevostamab, 2A10H7, 307307, 2A10D6, EPR27365-87, EPR26948-19, or EPR26948-67, or is disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558 each of which is incorporated by reference for all that they teach about anti-FCRL5 antibodies and their properties, or an antigen-binding portion thereof.
  • LRRC15 is a targeted cell surface antigen and binding moiety comprises the antigen binding domain of an anti-LRRC15 antibody.
  • the antigen binding domain of an anti-LRRC15 antibody is derived from samrotamab or DUNP19 or is disclosed in WO2005037999, WO2021022304, WO2024081729, WO2021102332, WO2021202642, WO2022157094, or WO2024158047, each of which is incorporated by reference for all that they teach about anti-LRRC15 antibodies and their properties, or an antigen-binding portion thereof.
  • a tLNP is targeted to a tumor cell.
  • the tumor cell expresses one of the antigens described above and the tLNP is targeted to antigen expressing tumors using the same means as described above.
  • the tLNP is targeted to some other tumor antigen, such as those enumerated in U.S. Provisional Application No.63/371,742, filed on August 17, 2022, entitled CONDITIONING FOR IN VIVO IMMUNE CELL ENGINEERING which is incorporated by reference for all that it teaches about the delivery of nucleic acids into tumor cells using tLNP that is not inconsistent with this disclosure.
  • the disclosed LNP and tLNP comprise a payload comprising or consisting of one or more nucleic acid species.
  • the LNP or tLNP payload comprises only one nucleic acid species while in other embodiments the LNP or tLNP payload comprises multiple nucleic acid species, for example, 2, 3, or 4 nucleic acid species.
  • the payload can comprise or consist of 1) a single nucleic acid species encoding a single species of CAR or ICE, 2) a single nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which each CAR and/or ICE has specificity for a same target antigen, 3) a single nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which at least one CAR and/or ICE has specificity for a different target antigen than the other(s), 4) two or more nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) in which each C
  • the nucleic acid can be RNA or DNA.
  • the nucleic acid can be multicistronic, for example, bicistronic.
  • LNPs or tLNPs of this disclosure further comprise a nucleic acid payload.
  • a nucleic acid is an mRNA, a self-replicating RNA (also known as self-amplifying RNA), a circular RNA, a siRNA, a miRNA, DNA, a gene editing component (for example, a guide RNA, a tracr RNA, an sgRNA), a gene writing component, an mRNA encoding a gene or base editing protein, a zinc-finger nuclease, a TALEN, a CRISPR nuclease, such as Cas9, a DNA molecule to be inserted or serve as a template for repair), and the like, or a combination thereof.
  • a gene editing component for example, a guide RNA, a tracr RNA, an sgRNA
  • a gene writing component for example, a guide RNA, a tracr RNA, an sgRNA
  • a gene writing component for example, a guide RNA, a tracr RNA, an sgRNA
  • the nucleic acid comprises small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO).
  • the nucleic acid comprises a self-replicating RNA or a circular RNA.
  • the mRNA encodes a reprogramming agent or comprises or encodes a conditioning agent.
  • the RNA linear mRNA, circular, or self-replicating
  • an mRNA encodes a chimeric antigen receptor (CAR).
  • an mRNA encodes a gene-editing or base-editing or gene writing protein.
  • a nucleic acid is a guide RNA.
  • an LNP or tLNP comprises both a gene- or base-editing or gene writing protein-encoding mRNA and one or more guide RNAs.
  • CRISPR nucleases can have altered activity, for example, modifying the nuclease so that it is a nickase instead of making double-strand cuts or so that it binds the sequence specified by the guide RNA but has no enzymatic activity.
  • Base-editing proteins are often fusion proteins comprising a deaminase domain and a sequence-specific DNA binding domain (such as an inactive CRISPR nuclease).
  • the reprogramming agent comprises an immune receptor (for example, a chimeric antigen receptor or a T cell receptor) or an immune cell engager (for example, a bispecific T cell engager (BiTE), a bispecific killer cell engager (BiKE), a trispecific kill cell engager (TriKE), a dual affinity retargeting antibody (DART), a TRIDENT (linking two DART units or a DART unit and a Fab domain), a macrophage engager (e.g., BiME), an innate cell engager, and the like).
  • the nucleic acid is an RNA, for example, mRNA, and the RNA comprises at least one modified nucleoside.
  • the modified nucleoside is pseudouridine, N 1 -methylpseudouridine, 5-methylcytosine, 5-methyluridine, N 6 - methyladenosine, 2’-O-methyluridine, or 2-thiouridine.
  • all of the uridines are substituted with a modified nucleoside. Further disclosure of modified nucleosides and their use can be found in U.S. Patent No.8,278,036 which is incorporated herein by reference for those teachings.
  • the reprogramming agent encodes or is a gene/genome editing component.
  • the gene/genome editing component is a guide RNA for an RNA-directed nuclease or other nucleic acid editing enzyme, clustered regularly interspaced short palindromic repeat RNA (crisprRNA), a trans-activating clustered regularly interspaced short palindromic repeat RNA (tracrRNA).
  • crisprRNA clustered regularly interspaced short palindromic repeat RNA
  • tracrRNA trans-activating clustered regularly interspaced short palindromic repeat RNA
  • the gene/genome editing component is a nucleic acid-encoded enzyme, such as RNA-guided nuclease, a gene or base editing protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, or a CRISPR nuclease (e.g., Cas9 or Cas 12, etc.).
  • the gene/genome editing component is DNA to be inserted or that serves as a template in gene or genome editing for example a template for repair of a double-strand break.
  • the nucleic acid can be multicistronic.
  • each agent or component is encoded or contained is a separate nucleic acid species.
  • two or more nucleic acid species are packaged together in a single LNP species.
  • a subset of the payload nucleic acid species to be delivered (e.g., a single nucleic acid species) is packaged in one LNP or tLNP species while another subset of the nucleic acid species is packaged in another LNP or tLNP species.
  • the different (t)LNP species can differ by only the payload they contain.
  • the different (t)LNP species can be combined in a single formulation or pharmaceutical composition for administration.
  • this disclosure provides a method of making a LNP or tLNP comprising mixing of an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of the lipids in proportions disclosed herein.
  • the mixing is rapid.
  • the aqueous solution is buffered at pH of about 3 to about 5, for example, without limitation, with citrate or acetate.
  • the alcohol can be ethanol, isopropanol, t-butanol, or a combination thereof.
  • the rapid mixing is accomplished by pumping the two solutions through a T-junction or with an impinging jet mixer.
  • Microfluidic mixing through a staggered herringbone mixer (SHM) or a hydrodynamic mixer (microfluidic hydrodynamic focusing), microfluidic bifurcating mixers, and microfluidic baffle mixers can also be used.
  • buffer for example phosphate, HEPES, or Tris
  • the diluted LNPs are purified either by dialysis or ultrafiltration or diafiltration using tangential flow filtration (TFF) against a buffer in a pH range of about 6 to about 8.5 (for example, phosphate, HEPES, or Tris) to remove the alcohol.
  • THF tangential flow filtration
  • the buffer is exchanged with like buffer containing a cryoprotectant (for example, glycerol or a sugar such as sucrose, trehalose, or mannose).
  • a cryoprotectant for example, glycerol or a sugar such as sucrose, trehalose, or mannose.
  • the LNPs are concentrated to a desired concentrated, followed by 0.2 ⁇ m filtration through, for example, a polyethersulfone (PES) or modified PES filter and filled into glass vials, stoppered, capped, and stored frozen.
  • PES polyethersulfone
  • a lyoprotectant is used and the LNP lyophilized for storage instead of as a frozen liquid.
  • WO2017/048770 each of which is incorporated by reference for all that they teach about the production of LNP.
  • Some aspects are a method of making a tLNP comprising rapid mixing of an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of the lipids as disclosed for LNP.
  • the lipid mixture includes functionalized PEG-lipid, for later conjugation to a targeting moiety.
  • functionalized PEG-lipid refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group (such as, maleimide, N-hydroxysuccinimide (NHS) ester, Cys, azide, alkyne, and the like) that can be used for conjugating a targeting moiety to the PEG-lipid, and thus, to the LNP comprising the PEG-lipid.
  • a chemically reactive group such as, maleimide, N-hydroxysuccinimide (NHS) ester, Cys, azide, alkyne, and the like
  • the functionalized PEG-lipid is inserted into and LNP subsequent to initial formation of an LNP from other components.
  • the targeting moiety is conjugated to functionalized PEG-lipid after the functionalized PEG-lipid containing LNP is formed.
  • the method comprises: i).
  • the method comprises: i).
  • the method comprises: i).
  • the method comprises: i).
  • tLNPs are purified by dialysis, tangential flow filtration, or size exclusion chromatography, and stored, as disclosed above for LNPs.
  • the encapsulation efficiency of the nucleic acid by the LNP or tLNP is typically determined with a nucleic acid binding fluorescent dye added to intact and lysed aliquots of the final LNP or tLNP preparation to determine the amounts of unencapsulated and total nucleic acid, respectively.
  • Encapsulation efficiency is typically expressed as a percentage and calculated as 100 x (T-U)/T where T is the total amount of nucleic acid and U is the amount of unencapsulated nucleic acid. In various embodiments, the encapsulation efficiency is ⁇ 80%, ⁇ 85%, ⁇ 90%, or ⁇ 95%.
  • a nucleic acid or other negatively charged payload
  • methods of delivering a nucleic acid (or other negatively charged payload) into a cell comprising contacting the cell with LNP or tLNP disclosed herein.
  • LNP or tLNP each of the herein disclosed genera, subgenera, and or species of LNP or tLNP disclosed herein including those based on the inclusion or exclusion of particular lipids, particular lipid compositions, particular payloads, and/or particular targeting moieties can be used in defining the scope of the methods of delivering a payload to a cell.
  • the contacting takes place ex vivo. In some embodiments, the contacting takes place extracorporeally.
  • the contacting takes place in vivo.
  • contacting in vivo can be accomplished through any appropriate route of administration.
  • an LNP or tLNP is contacted with target cells in vivo, by systemic or local administration.
  • the in vivo contacting comprises intravenous, intramuscular, subcutaneous, intralesional, intranodal or intralymphatic administration.
  • transfection of hepatocytes is reduced as compared to tLNPs comprising a conventional, prior art ionizable cationic lipid, such as ALC- 0315.
  • an LNP or tLNP is administered 1-3 times a week for 1, 2, 3, or 4 weeks.
  • toxicity is confined (or largely confined) to grades of 0 or 1 or 2, as discussed above.
  • Advantageous compact administration regimens for tLNPs for example, comprising one or more cycles of 2 or 3 administrations at 72 hour intervals, are described in U.S. Provisional Patent Application Number 63/556,735, filed February 22, 2024, U.S. Provisional Patent Application Number 63/708,513, filed October 17, 2024, U.S. Provisional Patent Application Number 63/721,154, filed November 15, 2024, and PCT patent application No. PCT/US2025/017092, filed February 24, 2025, which administration regimens are incorporated by reference in their entirety.
  • the herein disclosed LNP and tLNP compositions and formulations have reduced toxicity as compared to widely used prior LNP compositions such as those containing ALC- 0315.
  • the toxicity can be described as an observable toxicity, a substantial toxicity, a severe toxicity, or an acceptable toxicity, or a dose-limiting toxicity (such as but not limited to a maximum tolerated dose (MTD)).
  • MTD maximum tolerated dose
  • an observable toxicity it is meant that while a change is observed the effect is negligible or mild.
  • substantial toxicity it is meant that there is a negative impact on the patient’s overall health or quality of life. In some instances, a substantial toxicity can be mitigated or resolved with other ongoing medical intervention.
  • a severe toxicity it is meant that the effect requires acute medical intervention and/or dose reduction or suspension of treatment.
  • the acceptability of a toxicity will be influenced by the particular disease being treated and its severity and the availability of mitigating medical intervention.
  • toxicity is confined (or largely confined) to an observable toxicity.
  • toxicity is confined (or largely confined) to grades of 0 or 1 or 2.
  • the payload is a nucleic acid and the method of delivering is a method of transfecting.
  • the nucleic acid payload comprises an mRNA, circular RNA, self-amplifying RNA, or guide RNA.
  • Nucleic acid structures and especially mRNA structures, as well as individual RNA molecules encoding particular polypeptides, that are well-adapted to delivery by LNP or tLNP are disclosed in U.S. Provisional Patent Application Number.63/595,753 filed November 2, 2023, U.S. Provisional Patent Application Number.63/611,092 filed December 15, 2023, U.S. Provisional Patent Application Number 63/654,928, filed May 31, 2024, U.S. Provisional Patent Application Number 63/708,529, filed October 17, 2024, and International Patent Application No. PCT/US2024/054033, filed November 1, 2024, each of which is incorporated by reference for all that it teaches about nucleic acid payloads for in vivo transfection and their design.
  • the payload comprises a nucleic acid encoding an immune receptor or immune cell engager and the method of delivering is also a method of reprogramming an immune cell.
  • the payload comprises a nucleic acid that encodes, or is, a BRM and the method of delivering is also a method of providing a conditioning agent.
  • the BRM or conditioning agent is a gamma chain receptor cytokine such as IL-2, IL-7, IL-15, IL-15/15Ralpha, IL-21; an immune modulating (also known as pan activating) cytokine such as IL-12, IL-18; an immune checkpoint inhibitor such as an antagonist of cytotoxic T-lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), program cell death ligand 1 (PD-L1), T- cell immunoglobulin and mucin-domain-containing-3 (Tim-3), lymphocyte activation gene 3 (LAG-3) or indoleamine 2,3-dioxygenase (IDO) or agonists of 4-immunoglobulin and BB cell surface glycoprotein (4-1BB), OX40 or inducible costimulator (ICOS); a chemokine such as RANTES, IP10, MIG; or another BRM such as Flt3, GM-CSF, and G-CSF.
  • the payload comprises a nucleic acid encoding a gene/genome editing enzyme and/or a guide RNA or other component of a gene/genome editing system and the method of delivering is also a method of reprogramming a cell.
  • the cell is an immune cell.
  • the cell is an HSC.
  • the cell is an MSC.
  • the binding moiety binds to a lymphocyte surface molecule or a monocyte surface molecule.
  • Lymphocyte surface molecules include CD2, CD3, CD4, CD5, CD7, CD8, CD28, 4-1BB (CD137), CD166, CTLA-4, OX40, PD-1, GITR, LAG-3, TIM-3, CD25, low affinity IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, and IL-21 receptor.
  • Monocyte surface molecules include CD5, CD14, CD16a, CD32, CD40, CD11b (Mac-1), CD64, DEC205, CD68, and TREM2.
  • Exemplary antibodies that can provide antigen binding domains to bind these surface molecules are disclosed herein. Such antibodies, individually and collectively, constitute means for binding to an immune cell (or leukocyte) – or to a lymphocyte or monocyte, as indicated.
  • the binding moiety binds to a HSC surface molecule or a MSC surface molecule.
  • HSC surface molecules include CD117, CD34, CD44, CD90 (Thy1), CD105, CD133, BMPR2, and Sca-1.
  • MSC surface molecules include CD70, CD105, CD73, Stro-1, SSEA-4, CD271, CD146, GD2, SSEA-3, SUSD2, Stro-4, MSCA-1, CD56, CD200, PODXL, CD13, CD29, CD44, and CD10.
  • Exemplary antibodies that can provide antigen binding domains to bind these surface molecules are disclosed herein above.
  • this disclosure provides methods of treating a disease or disorder comprising administering a tLNP (of LNP) of this disclosure to a subject in need thereof.
  • a tLNP of LNP
  • a subject is a human.
  • a tLNP is administered systemically.
  • a tLNP is administered by intravenous or subcutaneous infusion or injection. In some embodiments, a tLNP is administered locally. In some embodiments, a tLNP is administered by intraperitoneal or intralesional infusion injection. [00384] In further embodiments, a tLNP can be administered in combination with the standard of care for a particular indication, such as corticosteroids (e.g., prednisone) for management of myositis or lupus nephritis.
  • corticosteroids e.g., prednisone
  • myositis is also treated with methotrexate, which can be combined with immunosuppressive agents (e.g., azathioprine, mycophenolate mofetil, tacrolimus), which are usually required in addition to corticosteroids.
  • immunosuppressive agents e.g., azathioprine, mycophenolate mofetil, tacrolimus
  • cyclical steroids and cyclophosphamide might be used in combination with tLNPs of this disclosure.
  • an anti-IL-6 such as tocilizumab
  • the disease or disorder is an autoimmune disease.
  • autoimmune disease examples include, without limitation, myocarditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, fibrosing alveolitis, multiple sclerosis, rheumatic fever, polyglandular syndromes, agranulocytosis, autoimmune hemolytic anemias, bullous pemphigoid, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, allergic responses, insulin-resistant diabetes, ps
  • the autoimmune disease is a T cell-mediated autoimmunity or a B cell-mediated autoimmunity.
  • the B cell-mediated autoimmune disease is myositis (such as anti-synthetase myositis), lupus nephritis, membranous nephropathy, systemic lupus erythematosus, anti-neutrophilic cytoplasmic antibody (ANCA) vasculitis, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, pemphigus vulgaris, rheumatoid arthritis, dermatomyositis, immune mediated necrotizing myopathy (IMNM), anti-synthetase syndrome, polymyositis, systemic sclerosis, diffuse cutaneous systemic sclerosis, limited cutaneous systemic sclerosis, anti-synthetase syndrome (idiopathic inflammatory myopathy
  • myositis such as anti-
  • the B cell-mediated autoimmune disease is myositis, lupus nephritis, membranous neuropathy, scleroderma, systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris.
  • the B cell-mediated autoimmune disease is myositis, lupus nephritis, membranous neuropathy, or scleroderma.
  • the B cell- mediated autoimmune disease is myositis. In some instances, the myositis is anti-synthetase myositis.
  • the B cell-mediated autoimmune disease is systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris.
  • the disease or disorder is rejection of an allogeneic organ or tissue graft.
  • Pre-existing antibodies and/or B cells, in their role as antigen presenting cells, can facilitate rapid immune rejection through known mechanisms hence depleting a large number of B cells can help prevent allograft rejection.
  • B cell depletion can be used in the management of graft-versus-host disease.
  • the disease or disorder is a graft-versus-host disease.
  • the disease or disorder is a cancer.
  • cancers include, without limitation, carcinomas, sarcomas, and hematologic cancers.
  • the hematologic cancer is a lymphoma, leukemia, or myeloma.
  • the hematologic cancer is a B lineage or T lineage cancer.
  • the B lineage cancer is multiple myeloma, diffuse large B cell lymphoma, acute myeloid leukemia, Mantle Cell lymphoma, follicular lymphoma, B acute lymphoblastic leukemia, chronic lymphocytic leukemia, or myelodysplastic syndrome.
  • the cancer is a sarcoma.
  • the cancer is a carcinoma, such as breast cancer, colon cancer, ovarian cancer, lung cancer, testicular cancer, or pancreatic cancer.
  • the cancer is melanoma.
  • Some embodiments are methods of B cell depletion utilizing a CAR or other reprogramming agent with specificity for a B cell antigen.
  • the term “B cell antigen” can refer to any antigen expressed by cell in the B cell lineage from pro-B cells through plasma cells.
  • Exemplary B cell antigens include CD19, CD20, CD22, BCMA, GPRC5D, and FCRL5, as well as antigens noted to be associated with hematologic cancers of the B cell lineage.
  • B cell-mediated autoimmunity, allotransplant rejection, GVHD, and the like as disclosed above B cell depletion blunt induction of anti-drug antibodies as an additional feature of such treatments or as an adjunct to other therapies.
  • the disease or disorder is a genetic disease or disorder such as a monogenic genetic disease.
  • the genetic disease or disorder is a hemoglobinopathy, for example, sickle cell disease or ⁇ -thalassemia.
  • the disease or disorder is a fibrotic disease or disorder.
  • a tLNP of this disclosure comprises a nucleic acid encoding a chimeric antigen receptor (CAR).
  • the receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor.
  • a nucleic acid encoding a CAR refers to one or more nucleic acid species encoding one or more CARs; for example, a single or multiple species of nucleic acid encoding a single CAR species, or multiple species of nucleic acid encoding multiple CAR species. In some instances, these multiple CAR species have a same specificity while in other instances they have multiple specificities.
  • a CAR of this disclosure is multispecific, for example, bispecific, comprising multiple antigen binding moieties each specific for separate antigens.
  • the CAR in LCAR-AIO targets three antigens — CD19, CD20 and CD22 (see, Blood (2021) 138 (Supplement 1): 1700).
  • a CAR can comprise an extracellular binding domain that specifically binds a target antigen, a transmembrane domain, and one or more intracellular signaling domains.
  • a CAR can further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, or one or more intracellular costimulatory domains. Domains can be directly adjacent to one another, or there can be one or more amino acids linking the domains.
  • the signal peptide can be derived from an antibody, a TCR, CD8 or other type 1 membrane proteins, preferably a protein expressed in a T or other immune cell.
  • the transmembrane domain can be one associated with any of the potential intracellular domains or from another type 1 membrane protein, such as TCR alpha, beta, or zeta chain, CD3 epsilon, CD4, CD8, or CD28, amongst other possibilities known in the art.
  • the transmembrane domain can further comprise a hinge domain located between the extracellular binding domain and the hydrophobic membrane-spanning region of the transmembrane domain.
  • the hinge domain and transmembrane domain are contiguous sequences in the same source protein.
  • the hinge and membrane-spanning domains are derived from CD28. In other instances, the hinge and membrane-spanning domains are derived from CD8 ⁇ .
  • the intracellular signaling domain can be derived from the CD3 zeta chain, DAP10, DAP12, Fc ⁇ RIII, FcsRI, or an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic domain, amongst other possibilities known in the art.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the intracellular costimulatory domain can be derived from CD27, CD28, 4-1BB, OX40, or ICOS, amongst other possibilities known in the art.
  • CARs are used to treat a disease or condition associated with a target cell that expresses the antigen targeted by the CAR.
  • an anti-CD19 or anti-CD20 CAR can be used to target and treat B cell malignancies or B cell-mediated autoimmune conditions or diseases (e.g., having an immune cell targeting moiety, such as an anti-CD8 antibody).
  • an anti- FAP CAR can be used to target and treat solid tumors or fibrosis (e.g., cardiac fibrosis, cancer-associated fibroblasts), which can also have an immune cell targeting moiety, such as an anti-CD8 antibody.
  • CARs that can be used in accordance with the embodiments described herein include to those disclosed in US 7,446,190, US 9,328,156, US 11,248,058, US20190321404, WO2019119822, WO2019159193, WO2020011706, WO2022125837, and WO2024086190 (anti-CD19), US 10,287,35 (anti-CD19), US 10,442,867 and US2021/0363245 (anti-CD19 and anti-CD20), US 10,543,263 (anti-CD22), WO2016149578 (anti-CD19 and anti-CD22), US 10,316,101, US 11,623,961 WO2015052538, WO2016166630, WO2017130223, WO2017173256, WO2019085102, WO2019241426, WO2020065330, WO2020038146, WO2020190737, WO2021091945 (anti-BCMA), WO2016130598 (anti-BCMA and syndecan-1), US 10,
  • Each CAR constitutes means for targeting an immune cell, for example, a T cell, to the indicated antigen.
  • Exemplary target antigens against which a CAR, TCR, or ICE can have specificity include, but are not limited to, B cell maturation agent (BCMA) ⁇ , CA9 ⁇ , CD4 ⁇ , CD5 ⁇ , CD19* ⁇ , CD20 (MS4A1)* ⁇ , CD22* ⁇ , FCRL5 ⁇ , GPRC5D ⁇ , CD23* ⁇ , CD30 (TNFRSF8)* ⁇ , CD33* ⁇ , CD38* ⁇ , CD44* , CD70* ⁇ , CD133 , CD174, CD274 (PD-L1)* ⁇ , CD276 (B7-H3) ⁇ , CEACAM5* ⁇ , CLL1 , CSPG4* , EGFR* ⁇ , EGFRvIII*, EPCAM* ⁇
  • Well-known TCR targets include NYESO-1, SSX2, PRAME, lineage specific antigens tyrosinase, PSMA, and Melan A/MART-1, and cancer testes antigens MAGE, GAGE, and LAGE.
  • * indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in US Patent 11,326,182B2 Table 9 or 10.
  • ⁇ indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in Wilkinson & Hale, 2022. Both references cited and incorporated by reference above.
  • the extracellular binding domain of the CAR comprises a ligand of a receptor expressed on the target cell.
  • the extracellular binding domain of the CAR comprises a ligand binding domain of a receptor for a ligand expressed on the target cell.
  • the advantages of the aspects and embodiments disclosed herein are independent of the specificity of the binding moiety. As such, the disclosed aspects and embodiments are generally agnostic to binding specificity. In certain embodiments, a particular binding specificity can be required.
  • the tLNP comprises a nucleic acid encoding an anti-CD19 chimeric antigen receptor (CAR).
  • the nucleic acid comprises mRNA. Examples of anti-CD19 CARs include those incorporating a CD19 binding moiety derived from the human antibody 47G4 or the mouse antibody FMC63.
  • the anti-CD19 CAR is the CAR found in tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, or brexucabtagene autoleucel.
  • Each of these CARs constitutes means for targeting an immune cell, for example, a T cell, to CD19.
  • the entire contents of each of foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, and activity of anti-CD19 CARs.
  • certain embodiments include tLNPs encapsulating a CD19 CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody.
  • the tLNP comprises a nucleic acid encoding an anti-CD20 chimeric antigen receptor (CAR).
  • CD20 is an antigen found on the surface of B cells as early as the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkin's disease, myeloma, and thymoma.
  • the nucleic acid comprises mRNA.
  • anti-CD20 CARs include those incorporating a CD20 binding moiety derived from an antibody specific to CD20, including, for example, Leu16, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab.
  • the anti-CD20 CAR is derived from a CAR specific to CD20, including, for example, MB-106 (Fred Hutchinson Cancer Research Center, see Shadman et al., Blood 134(Suppl.1):3235 (2019)) UCART20 (Cellectis, www.cellbiomedgroup.com), or C-CAR066 (Cellular Biomedicine Group, see Liang et al., J. Clin. Oncol.39(15) suppl:2508 (2021)).
  • MB-106 Red Hutchinson Cancer Research Center, see Shadman et al., Blood 134(Suppl.1):3235 (2019)
  • UCART20 Cellbiomedgroup.com
  • C-CAR066 Cellular Biomedicine Group, see Liang et al., J. Clin. Oncol.39(15) suppl:2508 (2021)).
  • the extracellular binding domain of the anti-CD20 CAR comprises an scFv derived from the Leu16 monoclonal antibody, which comprises the heavy chain variable region (V H ) and the light chain variable region (V L ) of Leu16 connected by a linker.
  • V H heavy chain variable region
  • V L light chain variable region
  • certain embodiments include tLNPs encapsulating a CD20 CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody.
  • the tLNP comprises a nucleic acid encoding an anti- BCMA chimeric antigen receptor (CAR).
  • CAR anti- BCMA chimeric antigen receptor
  • BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity.
  • the expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and non- Hodgkin's lymphoma, various leukemias, and glioblastoma.
  • the nucleic acid comprises mRNA.
  • anti-BCMA CARs include those incorporating a BCMA binding moiety derived from C11D5.3, a Mouse monoclonal antibody as described in Carpenter et al., Clin. Cancer Res.19(8):2048-2060 (2013). See also PCT Application Publication No. WO 2010/104949.
  • the extracellular binding domain of the BCMA CAR comprises an scFv derived from another Mouse monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res.19(8):2048-2060 (2013) and PCT Application Publication No. WO2010104949.
  • the extracellular binding domain of the BCMA CAR comprises an scFv derived from a Mouse monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther.29(5):585-601 (2016). See also, PCT Application Publication No. WO2012163805.
  • the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol.11(1):141 (2016), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO 2018/028647.
  • the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11(1):283 (2020), also referred to as FHVH33. See also, PCT Application Publication No. WO 2019/006072.
  • the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No.11,026,975 B2.
  • Further anti-BCMA CARs are disclosed in U.S. Application Publication Nos.2020/0246381 and 2020/0339699. Further anti-BCMA CARs include Allo-605 (described in U.S. Patent Publication No.20200261503), CT053 (described in U.S. Patent No. US11,525,006), Descartes-08 (described in U.S. Patent No.10,934,337), LCAR-B38M (described in U.S.
  • Patent No.10,934,363 PersonGen anti-BCMA CAR (described in CN114763383), Pregene Bio anti-BCMA CAR (described in U.S. Patent Publication No. US20220218746), the CAR in ciltacabtagene autoleucel (binding moiety described in US20170051068), and the CAR in idecabtagene vicleucel (described in U.S. Patent No. 10,383,929).
  • Each of these CARs constitutes means for targeting an immune cell, for example, a T cell, to BCMA.
  • Further antibodies comprising an anti-BCMA antigen binding domains that can be used in construction a CAR include AMG224 (described with other anti- BCMA antibodies in U.S.
  • Patent No.9,243,058), EMB-06 (described with other anti-BCMA antibodies in U.S. Patent Publication No. US20230002489), HPN217(described in U.S. Patent No.11,136,403), MEDI2228 (described in U.S. Patent No.10,988,546), REGN5459 (described in U.S. Patent No.11,384,153), SAR445514 (described in U.S. Patent Publication No.20240034816), SEA-BCMA (described in U.S. Patent No.11,078,291), TNB- 383B (described in U.S. Patent No.11,505,606), TQB2934 (described in U.S.
  • Patent Publication No.20230193292 WV078 (described in U.S. Patent No.11,492,409), alnuctamab (described in U.S. Patent No.10,683,369), belantamab (described in U.S. Patent No.9,273,141), elranatamab (described in U.S. Patent No.11,814,435), ispectamab (described in U.S. Patent Publication No.20210130483), linvoseltamab (described in U.S. Patent No.11,919,965), pavurutamab (described in U.S.
  • Patent No.11,419,933), and teclistamab (described in U.S. Patent No.10,072,088).
  • the entire contents of each of foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, and activity of anti-BCMA CARs and anti-BCMA antibodies that can provide an antigen binding domain for a CAR or immune cell engager.
  • certain embodiments include tLNPs encapsulating a BCMA CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti- CD8 antibody.
  • the tLNP comprises a nucleic acid encoding an anti- GPRC5D chimeric antigen receptor (CAR).
  • CAR anti-GPRC5D chimeric antigen receptor
  • GPRC5D is a G protein-coupled receptor without known ligands and of unclear function in human tissue. However, this receptor is expressed in myeloma cell lines and in bone marrow plasma cells from patients with multiple myeloma. GPRC5D has been identified as an immunotherapeutic target in multiple myeloma and Hodgkin lymphomas.
  • anti-GPRC5D CARs examples include those incorporating a GPRC5D binding moiety such as MCARH109 (Mailankody et al., N Engl J Med.387(13): 1196-1206 (2022)), BMS-986393, or OriCAR-017 (Rodriguez-Otero et al., Blood Cancer J. 14(1): 24 (2024)).
  • anti-GPRC5D CARs include those incorporating a GPRC5D binding moiety derived from an antibody specific to GPRC5D, for example, talquetamab (Pillarisetti et al., Blood 135:1232-43 (2020)), or forimtamig.
  • the extracellular binding domain of the anti-GPRC5D CAR comprises an scFv derived from a 6D9 Mouse antibody with specificity to human GPRC5D (see creative-biolabs.com/car-t/anti- gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm).
  • the extracellular binding domain of the GPRC5D CAR comprises an scFv of anti-GPRC5D antibody linked to 4-1BB or CD28 costimulatory domain and CD3 ⁇ signaling domain as described in Mailankody et al., N Engl J Med.387(13): 1196-1206 (2022); creative-biolabs.com/car-t/anti- gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm; and Rodriguez-Otero et al., Blood Cancer J.14(1): 24 (2024).
  • tLNPs encapsulating an anti-GPRC5D CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody.
  • the tLNP comprises a nucleic acid encoding an anti- FCRL5 chimeric antigen receptor (CAR).
  • FCRL5 Fc receptor-like 5
  • FCRH5 Fc receptor-like 5
  • BXMAS1, CD307, CD307E, and IRTA2 is a protein marker expressed on the surface of plasma cells in patients with multiple myeloma. Furthermore, contact with FCRL5 stimulates B-cell proliferation; thus, FCRL5 has been identified as an immunotherapeutic target for this disease.
  • anti-FCRL5 CARS include those incorporating an FCRL5 binding moiety, such as those described in WO2016090337, WO2017096120, WO2022263855, and WO2024047558.
  • the extracellular binding domain of the anti-FCRL5 CAR comprises an scFv with specificity to FCRL5, such as ET200-31, ET200-39, ET200-69, ET200-104, ET200-105, ET200-109, or ET200-117.
  • the extracellular binding domain of the anti-FCRL5 CAR comprises an scFv derived from a mouse antibody with specificity to human FCRL5.
  • Such antibodies include 7D11, F25, F56, and F119, as described in Polson et al., Int. Immunol., 18(9): 1363-1373 (2006); Franco et al., J. Immunol.
  • the extracellular binding domain of the anti-FCRL5 CAR comprises a binding moiety derived from the antigen binding domain of an anti-FCRL5 antibody or nanobody, including cevostamab, 2A10H7, 307307, 2A10D6, 13G9, 10A8, 509f6, EPR27365-87, EPR26948-19, or EPR26948-67, or as disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558.
  • the extracellular binding domain of the anti-FCRL5 CAR comprises a binding moiety derived from an antibody-drug conjugate targeting FCRL5, such as those described in Elkins et al., Mol.
  • the extracellular binding domain of the anti-FCRL5 CAR is linked to a costimulatory domain, such as a 4-1BB or CD28 costimulatory domain, and a signaling domain, such as a CD3 ⁇ signaling domain.
  • a costimulatory domain such as a 4-1BB or CD28 costimulatory domain
  • a signaling domain such as a CD3 ⁇ signaling domain.
  • certain embodiments include tLNPs encapsulating a FCRL5 CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody.
  • the tLNP comprises nucleic acids encoding one or more CARs that target one or more multiple antigens.
  • the tLNP comprises distinct mRNAs that are encapsulated together in a single tLNP, with each mRNA encoding one monospecific CAR.
  • the tLNP can comprise an mRNA encoding an anti- CD19 CAR and an mRNA encoding an anti-CD20 CAR, an mRNA encoding an anti-CD19 CAR and an mRNA encoding an anti-BCMA CAR, an mRNA encoding an anti-GPRC5D CAR and an mRNA encoding an anti-BCMA CAR, or an mRNA encoding an anti-FCRL5 CAR and an mRNA encoding an anti-BCMA CAR.
  • the tLNP comprises a single mRNA encoding a bicistronic mRNA encoding two monospecific CARs.
  • the bicistronic mRNA can encode an anti-CD19 CAR and an anti-CD20 CAR, an anti-CD19 CAR and an anti-BCMA CAR, an anti-GPRC5D CAR and an anti-BCMA CAR, or an anti-FCRL5 CAR and an anti-BCMA CAR.
  • the tLNP comprises a single mRNA encoding an mRNA encoding a multispecific CAR. In some embodiments, the tLNP comprises a single mRNA encoding an mRNA encoding a bispecific CAR.
  • the mRNA can encode an anti-CD19 and anti-CD20 bispecific CAR, an anti-CD19 and anti-BCMA bispecific CAR, an anti-GPRC5D and anti-BCMA bispecific CAR, or an anti- FCRL5 and anti-BCMA bispecific CAR.
  • multiple tLNPs can be co- formulated in a combination with each comprising one mRNA encoding one monospecific CAR.
  • two tLNPs can be co-formulated with one tLNP comprising an mRNA encoding an anti-CD19 CAR and the other tLNP comprising an mRNA encoding an anti- CD20 CAR, one tLNP comprising an mRNA encoding an anti-C19 CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR, one tLNP comprising an mRNA encoding an anti-GPRC5D CAR and the other tLNP comprising an mRNA encoding an anti- BCMA CAR, or one tLNP comprising an mRNA encoding an anti-FCRL5 CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR.
  • multiple tLNPs can be co-administered in a combination, either simultaneously or sequentially, wherein each comprises one mRNA encoding one monospecific CAR.
  • two tLNPs can be co-administered in a combination, either simultaneously or sequentially, with one tLNP comprising an mRNA encoding an anti-CD19 CAR and the other tLNP comprising an mRNA encoding an anti-CD20 CAR, one tLNP comprising an mRNA encoding an anti- C19 CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR, one tLNP comprising an mRNA encoding an anti-GPRC5D CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR, or one tLNP comprising an mRNA encoding an anti- FCRL5 CAR and the other tLNP comprising an mRNA encoding an encoding an
  • the targeting can be mediated by any of the CARs described herein which constitute means for targeting cells expressing the indicated antigen.
  • Cellular therapy involving the administration of genetically engineered cells to a patient has generally required depleting or ablative conditioning to facilitate engraftment of the engineered cells (for example, T cells or HSC).
  • depleting or ablative conditioning to facilitate engraftment of the engineered cells (for example, T cells or HSC).
  • T cells or HSC for example, T cells or HSC.
  • conditioning agents include biological response modifiers (BRMs) that can be delivered directly to a subject or encoded in nucleic acid molecules, including as mRNA, and delivered to a subject using the LNP and tLNP compositions and formulations disclosed herein.
  • BRMs biological response modifiers
  • an encoded conditioning agent comprises a ⁇ -chain receptor agonist, an inflammatory chemokine, a pan-activating cytokine, an antigen presenting cell activity enhancer, an immune checkpoint inhibitor, or an anti-CCR4 antibody.
  • the ⁇ -chain receptor cytokine comprises IL-15, IL-2, IL-7, or IL-21.
  • the immune checkpoint inhibitor comprises an anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-Tim-3, or anti-LAG-3 antibody.
  • the inflammatory chemokine comprises CCL2, CCL3, CCL4, CCL5, CCL11, CXCL1, CXCL2, CXCL-8, CXCL9, CXCL10, or CXCL11.
  • the antigen presenting cell activity enhancer comprises Flt-3 ligand, gm-CSF, or IL-18.
  • a pan-activating cytokine comprises IL-12 of IL 18.
  • a conditioning agent comprises a transcription factor, for example, one selected from the group consisting of nuclear factor of activated T-cells (NFAT), NF- ⁇ B, T-bet, signal transducer and activator of transcription 4 (STAT4), Blimp-1, c-Jun, and Eomesodermin (Eomes) and the tLNP is targeted to a T cell.
  • NFAT nuclear factor of activated T-cells
  • STAT4 signal transducer and activator of transcription 4
  • Blimp-1 Blimp-1
  • c-Jun c-Jun
  • Eomes Eomes
  • a tLNP encapsulating the nucleic acid-encoded conditioning agent is administered systemically, for example, by intravenous or subcutaneous infusion or injection.
  • the tLNP is administered locally, for example, by intralesional or intraperitoneal injection or infusion.
  • nucleic acid molecules encoding the conditioning agent and the engineering agent are encapsulated in the same tLNP while in other embodiments they are encapsulated in separate tLNPs. These two modes of delivery of conditioning agents are described in greater detail in PCT application PCT/US 2023/072426, which is incorporated by reference for all that it teaches about conditioning agents and their delivery of LNPs or tLNPs that is not inconsistent with this disclosure.
  • the nucleic acid comprises mRNA.
  • treating broadly includes any kind of treatment activity, including the mitigation, cure or prevention of disease, or aspect thereof, in man or other animals, or any activity that otherwise affects the structure or any function of the body of man or other animals.
  • Treatment activity includes the administration of the medicaments, dosage forms, and pharmaceutical compositions described herein to a patient, especially according to the various methods of treatment disclosed herein, whether by a healthcare professional, the patient his/herself, or any other person.
  • Treatment activities include the orders, instructions, and advice of healthcare professionals such as physicians, physician’s assistants, nurse practitioners, and the like, that are then acted upon by any other person including other healthcare professionals or the patient him/herself.
  • the orders, instructions, and advice aspect of treatment activity can also include encouraging, inducing, or mandating that a particular medicament, or combination thereof, be chosen for treatment of a condition - and the medicament is actually used - by approving insurance coverage for the medicament, denying coverage for an alternative medicament, including the medicament on, or excluding an alternative medicament, from a drug formulary, or offering a financial incentive to use the medicament, as might be done by an insurance company or a pharmacy benefits management company, and the like.
  • treatment activity can also include encouraging, inducing, or mandating that a particular medicament be chosen for treatment of a condition - and the medicament is actually used - by a policy or practice standard as might be established by a hospital, clinic, health maintenance organization, medical practice or physicians’ group, and the like. All such orders, instructions, and advice are to be seen as conditioning receipt of the benefit of the treatment on compliance with the instruction.
  • a financial benefit is also received by the patient for compliance with such orders, instructions, and advice.
  • a financial benefit is also received by the healthcare professional for compliance with such orders, instructions, and advice.
  • Some embodiments of these methods of treatment comprise administration of an effective amount of a compound or a composition disclosed herein.
  • a therapeutically effective amount is not necessarily a clinically effective amount, that is, while there can be therapeutic benefit as compared to no treatment, a method of treatment may not be equivalent or superior to a standard of care treatment existing at some point in time.
  • Other instances relate to a pharmacologically effective amount, that is an amount or dose that produces an effect that correlates with or is reasonably predictive of therapeutic (or prophylactic) utility.
  • therapeutically effective amount is synonymous with “therapeutically effective dose” and means at least the minimum dose of a compound or composition disclosed herein necessary to achieve the desired therapeutic or prophylactic effect.
  • a pharmacologically effective dose means at least the minimum dose of a compound or composition disclosed herein necessary to achieve the desired pharmacologic effect. Some embodiments refer to an amount sufficient to prevent or disrupt a disease process, or to reduce the extent or duration of pathology. Some embodiments refer to a dose sufficient to reduce a symptom associated with the disease or condition being treated. [00405]
  • the following examples are intended to illustrate various embodiments. As such, the specific embodiments discussed are not to be constructed as limitations on the scope of this disclosure. It is apparent to one skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
  • the mixture was stirred for 18 hours at 25°C, the was diluted with n-heptane (500mL) and the resulting solution was washed with water (200mL).
  • the organic phase was separated silica gel (50g, type ZCX-2, 100-200 mesh) was added and the solvent was removed in vacuo.
  • the dry, reaction impregnated silica gel was placed atop a column of silica gel (500g, type ZCX- 2, 100-200 mesh) in a Combi-flash apparatus. The column was eluted with a gradient of n- heptane/EtOAc from 100:0 to 95:5.
  • Step 2 A solution of the t-butyl ester of step 1 (24.0g, 51.0mmol) in toluene (110 mL) was cooled to 5°C under nitrogen, then CF 3 CO 2 H (53.64g, 0.47mol, 36mL) was added dropwise over 30 minutes. The mixture was allowed to warm to room temperature and was stirred at room temperature for 18 hours. The solution was diluted with n-heptane (370mL) and then 10% aq. K 2 HPO 4 solution (500mL) was added, and the mixture was stirred for 10 minutes.
  • the organic phase was separated, and the aqueous phase was extracted with n- heptane (2x250mL).
  • the aqueous phase was acidified by the addition of 1.2M aq. HCl (250mL) and the solution was extracted with n-heptane (500mL).
  • the organic phase was washed with an aqueous methanol solution (1:1, 250mL) and then was dried over anhydrous Na 2 SO 4 . Filtration and concentration in vacuo afforded acid 2 (18.0g, 43.4mmol, 85% yield) as a pale-yellow oil.
  • Example 2 Synthesis of O' 1 ,O 1 -(((tert-Butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): 4 [00410] To a solution of 2 (8.08g, 19.51mmol), in acetonitrile (160mL) under nitrogen at 25°C, was added in order diol 3 (2.00g, 9.76mmol), DMAP (1.19g, 9.76mmol) and EDCl (4.68g, 24.39mmol).
  • Example 3 Synthesis of bis(2-((5-(Octyloxy)-3-(2-(octyloxy)-2-oxoethyl)-5- oxopentanoyl) oxy)ethyl)ammonium trifluoroacetate: 5 [00412] To a solution of 4 (9.00g, 9.02mmol), in dichloromethane (DCM, 45mL) under nitrogen at 25°C, was added CF 3 CO 2 H (20.11g, 176mmol, 13.5mL) over a period of 10 minutes. The mixture was allowed to stir for 3 hours at 25°C, then was concentrated in vacuo and the residue was dissolved in n-heptane (400mL).
  • DCM dichloromethane
  • Example 5 Synthesis of O' 1 ,O 1 -(((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1- diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-250 [00416] To a solution of 6 (8.50g crude, 87.5% HCLC purity) in acetonitrile (85mL), cooled to 0°C under nitrogen, was added methyl trifluoromethanesulfonate (MeOTf, 1.55g, 9.43mmol) over a period of 5 minutes.
  • MeOTf methyl trifluoromethanesulfonate
  • Example 6 Synthesis of O'1,O1-((((2-(Dimethylamino)ethoxy)carbonyl) azanediyl) bis(ethane-2,1-diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL- 250 [00418] 3-(Carboxymethyl)pentanedioic acid 1 (Chem. Eur. J.2010, 16, 4037; Org. Chem. Frontiers 2017, 4, 1819) is coupled with 1-octanol, utilizing EDC-HCl and DMAP in CH 3 CN to give 2.
  • Example 7 Synthesis of O'1,O1-(2-(((2-(dimethylamino)ethoxy)carbonyl) amino)propane-1,3-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-291 [00419]
  • the coupling of diester 2 with diol 7 (Combi-Blocks #QI-4068), utilizing EDC-HCl and DMAP in CH 3 CN, results in 8.
  • Deprotection of the BOC-amine 8 with CF 3 CO 2 H in CH 2 Cl 2 , provides ammonium trifluoroacetate salt 9.
  • Example 8 Synthesis of O'1,O1-(2-(2-(2-(dimethylamino)ethoxy)-2-oxoethyl)propane- 1,3-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-292 [00420]
  • diol 11 Org. Proc. Res. Dev.2011, 15, 515
  • EDC-HCl and DMAP in CH 3 CN
  • Example 10 Synthesis of O'1,O1-(3-(3-(dimethylamino)propanamido)pentane-1,5-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-294
  • the coupling of diester 2 with diol 17 (Org. Proc. Res. Dev.2009, 13, 428), utilizing EDC-HCl and DMAP in CH 3 CN, results in 18.
  • Deprotection of the BOC-amine 18, with CF 3 CO 2 H in CH 2 Cl 2 provides ammonium trifluoroacetate salt 19.
  • Dev.2009, 13, 478) provides amine 22 which leads to amino diol 23 after reduction with LiAlH 4 in THF or diethyl ether. Hydrogenolysis (H 2 , Pd/C) of 23 in the presence of BOC-anhydride then affords N-protected amino diol 24.
  • the coupling of 24 with acid 2 (EDC-HCl, DMAP, Et 3 N in CH 2 Cl 2 , THF, or CH 3 CN) provides BOC-protected amine 25 which yields the ammonium trifluoroacete salt 26 after BOC removal (CF 3 CO 2 H in CH 2 Cl 2 or toluene).
  • Amine salt 26 then couples (EDC-HCl, DMAP, Et 3 N in CH 2 Cl 2 , THF, or CH 3 CN) with 3-dimethylamino propionic acid to prepare CICL-295.
  • Example 12 Synthesis of O'6,O6-(2-((2-(dimethylamino)ethoxy)carbonyl)-2- methylpropane-1,3-d iyl) 1,1'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)hexanedioate): CICL-296.
  • Example 15 Synthesis of Tetraoctyl 2,2',2'',2''-((((2-(2-(2-(2-(dimethylamino) ethoxy)-2- oxoethyl)propane-1,3-diyl)bis(oxy))bis(2-oxoethane-2,1- diyl))bis(azanetriyl))tetraacetate: CICL-299 [00427] The coupling of diester 40 with diol 11, utilizing EDC-HCl and DMAP in CH 3 CN, results in 47. Deprotection of the t-butyl ester 47, with CF 3 CO 2 H in CH 2 Cl 2 , provides acid 48. The coupling of acid 48 with N,N-dimethylaminoethanol, using HATU and i-Pr 2 NET in THF, leads to CICL-299.
  • Example 16 Synthesis of Tetraoctyl 2,2',2'',2''-((((3-((3-(dimethylamino)propanoyl)oxy) pentane-1,5-diyl)bis(oxy))bis(2-oxoethane-2,1-diyl))bis(azanetriyl))tetraacetate: CICL- 300
  • the coupling of alcohol 50 with N,N-dimethyl- ⁇ -alanine, using EDC-HCl, DMAP, and Et 3 N in CH 2 Cl 2 leads to CICL-300.
  • Example 17 Synthesis of Tetraoctyl 2,2',2'',2''-((((3-(3-(dimethylamino)propanamido) pentane-1,5-diyl)bis(oxy))bis(2-oxoethane-2,1-diyl))bis(azanetriyl))tetraacetate: CICL- 301 [00429]
  • Deprotection of the BOC-amine 51, with CF 3 CO 2 H in CH 2 Cl 2 provides ammonium trifluoroacetate salt 52.
  • Example 19 Synthesis of Tetraoctyl 2,2',2'',2''-((((2-((2- (dimethylamino)ethoxy)carbonyl)-2-methylpropane-1,3-diyl)bis(oxy))bis(3- oxopropane-3,1-diyl))bis(azanetriyl))tetraacetate: CICL-303 [00431] Hydrolysis of the bis-t-butyl esters present in 55 (Bioorg. Med. Chem.
  • Example 20 Synthesis of O' 1 ,O 1 -(((((1-methylazetidin-3-yl)oxy)carbonyl)azanediyl)bis (ethane-2,1-diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-250- 61 [00432] Acylimidazolide 6, when reacted with MeOTf provides an activated acylimidazolium species.
  • Example 23 Synthesis of 4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butanoic acid 79 [00437] To a solution of 78 (90.0g, 96.3% purity, 0.184mol) in toluene (0.41L), cooled in an ice-water bath under nitrogen, was added TFA (208.46g, 1.83mol, 140mL) over a period of 30 minutes. After the addition was complete the mixture was warmed to 15°C and the mixture was stirred for 18 hours. The chilled solution was cast into n-heptane (1.50L) and the resulting solution was extracted with 5% aq. potassium phosphate (2.0L) and the aqueous phase was collected.
  • TFA 208.46g, 1.83mol, 140mL
  • Example 24 Synthesis of O'1,O1-((((2-(dimethylamino)ethoxy)carbonyl)azanediyl) bis(ethane-2,1-diyl)) 5,5'-dioctyl bis(3-((nonanoyloxy)methyl)pentanedioate): CICL-309
  • Example 25 Synthesis of 1-(2-(((2-(dimethylamino)ethoxy)carbonyl)(2-((3- ((nonanoyloxy)methyl)-5-(octyloxy)-5-oxopentanoyl)oxy)ethyl)amino)ethyl) 5-octyl 3- (2-(octyloxy)-2-oxoethyl)pentanedioate: CICL-310 [00440] The coupling of acid 83 (1 equiv.) with diol 3 affords mono-esterified alcohol 87 after purification to remove unreacted 3 and bis-esterified 84.
  • Alcohol 87 is coupled with acid 2 utilizing EDCl and DMAP in CH 3 CN, which results in 88.
  • Deprotection of the BOC-blocked amine with TFA in CH 2 Cl 2 provides ammonium trifluoracetate salt 89.
  • Treatment of 89 with carbonyl-diimidazole and Et 3 N in CH 2 Cl 2 results in acyl-imidazole 90.
  • Alkylation of 90 with MeOTf in CH 3 CN, followed by the reaction of the derived acyl-imidazolium salt with 2- dimethylaminoethanol and (CH 3 ) 3 N results in CICL-310.
  • Example 26 Synthesis of 1-(2-(((2-(dimethylamino)ethoxy)carbonyl)(2-((4- (nonanoyloxy)-3-((nonanoyloxy)methyl)butanoyl)oxy)ethyl)amino)ethyl) 5-octyl 3- ((nonanoyloxy) methyl)pentanedioate: CICL-311 93 CICL-311 [00441] Alcohol 87 is coupled with acid 79 utilizing EDCl and DMAP in CH 3 CN, which results in 91. Deprotection of the BOC-blocked amine with TFA in CH 2 Cl 2 provides ammonium trifluoracetate salt 92.
  • An identical analytical paradigm starting with calculated pKa, can be performed with the head groups presented in Table 3 to select candidates appropriate for measured pKa alteration for the frameworks of CICL-250, CICL-291, CICL-292, CICL-296, CICL-297, CICL-298, CICL-299, CICL-303, CICL-309, CICL-310, and CICL-311.
  • Example 27 An identical analytical paradigm, starting with calculated pKa, can be performed with the head groups presented in Table 3 to select candidates appropriate for measured pKa alteration for the frameworks of CICL-250, CICL-291, CICL-292, CICL-296, CICL-297, CICL-298, CICL-299, CICL-303, CICL-309, CICL-310, and CICL-311.
  • tLNP incorporating CICL-250, or for comparison, CICL-1, and conjugated to an anti-mouse CD5 antibody were prepared and administered to C57BL/6 mice.
  • the tLNP comprised either CICL-250 or CICL-1, DSPC, cholesterol, DSG-PEG(2000), and DSPE-PEG(2000)-maleimide in the proportions indicated in Table 5 (below) and an N/P ratio of 6.
  • CICL-1 has the structure: [00445] Briefly, to prepare the tLNP, N1-methylpseudouridine (m1 ⁇ )-substituted mCherry mRNA (SEQ ID NO: 2) was encapsulated in LNP using a self-assembly process in which an aqueous solution of mRNA at pH 3.5 was rapidly mixed with a solution of lipids dissolved in ethanol, then followed by stepwise phosphate and Tris buffer dilution and tangential flow filtration (TFF) purification. Then an anti-CD5 mAb was conjugated to the above LNP to generate tLNP.
  • m1 ⁇ N1-methylpseudouridine
  • SEQ ID NO: 2 N1-methylpseudouridine (m1 ⁇ )-substituted mCherry mRNA
  • rat anti-mouse CD5 antibody clone 53-7.3 (BioLegend) was coupled to LNP via N-succinimidyl S-acetylthioacetate (SATA)–maleimide conjugation chemistry.
  • SATA N-succinimidyl S-acetylthioacetate
  • LNPs with DSPE-PEG(2000)-maleimide incorporated were formulated and stored at 4°C on the day of conjugation.
  • the antibody was modified with SATA (Sigma-Aldrich) to introduce sulfhydryl groups at accessible lysine residues allowing conjugation to maleimide.
  • SATA was deprotected using 0.5 M hydroxylamine followed by removal of the unreacted components by G-25 Sephadex Quick Spin Protein columns (Roche Applied Science, Indianapolis, IN).
  • the reactive sulfhydryl group on the antibody was then conjugated to maleimide moieties on the LNPs using thioether conjugation chemistry.
  • Purification was performed using Sepharose CL-4B gel filtration columns (Sigma-Aldrich).
  • tLNPs LNPs conjugated with a targeting antibody
  • tLNPs were frozen at -80°C.
  • the particle size (hydrodynamic diameter) and polydispersity index of the targeted lipid nanoparticles were determined using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Size measurement was carried out in pH 7.4 Tris buffer at 25°C in disposable capillary cells.
  • NIBS non-invasive back scatter system
  • mRNA content was determined using a Quant-iT TM RiboGreen RNA assay kit (Invitrogen TM ).
  • tLNP antibody to mRNA weight ratio was determined with the BCA (bicinchoninic acid) total protein assay and Ribogreen® assay of mRNA content. Table 5. Physicochemical properties of the tLNP [00447] As seen in Table 5, all of these tLNP compositions had hydrodynamic diameters and polydispersity indices within the acceptable ranges of 50-150 nm and ⁇ 0.2 for PDI. Encapsulation efficiency is acceptable at ⁇ 80% although ⁇ 85% and ⁇ 90% are preferred. Binder density (Ab:mRNA ratio (wt:wt)) is acceptable at ratios of 0.3 to 1.0.
  • the apparent or measured pKa of ionizable lipid in the lipid nanoparticle was determined using 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS salt, Toronto Research Chemicals, Toronto, ON, Canada). Lipid nanoparticles were diluted in 1xDulbecco's PBS to a concentration of 1 mM total lipids. TNS salt was prepared as a 1 mg/mL stock solution in DMSO and then further diluted using distilled water to a working solution of 60 ⁇ g/mL (179 mM).
  • Diluted lipid nanoparticle samples were further diluted to 90 ⁇ M total lipids in 165 ⁇ L of buffered solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, and final TNS concentration of 1.33 ⁇ g/mL (4 ⁇ M) with the pH ranging from 3.5 to 12.2.
  • fluorescence intensity was measured at room temperature in a BioTek Synergy H1 plate reader using excitation and emission wavelengths of 321 and 445 nm, respectively.
  • the fluorescence signal was blank subtracted and plotted as a function of the pH, then analyzed using a nonlinear (Boltzmann) regression analysis with the apparent pKa determined as the pH giving rise to half maximal fluorescence intensity as calculated by the Henderson ⁇ Hasselbalch equation.
  • the mice were administered tLNPs containing 10 ⁇ g of mRNA by tail vein injection and tissue was harvested 24 hours later.
  • tLNP comprising DSG- PEG(2000) and either CICL-250 or CICL-1 had similar performance in splenic T cells for both transfection rate and expression level (measured as molecular equivalent of soluble fluorochrome (MESF)).
  • the tLNP incorporating CICL-250 and CICL-1 had similarly low transfection rates ( ⁇ 4%) for CD45- liver cells (hepatocytes) ( Figure 1B).
  • the tLNP incorporating CICL-250 and CICL-1 had similarly low levels in liver Kupffer cells (CD45 + /CD11 + liver cells) ( Figure 1C).
  • Additional aspects of the disclosure are provided by the following enumerated embodiments, which can be combined in any number and in any combination not technically or logically inconsistent. This is not an exhaustive listing of the embodiments disclosed which include similar embodiments directed to difference species and genera than those exemplified her. Furthermore, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of this invention.
  • each R 1 is independently a C 7 -C 11 alkyl or a C 7 -C 11 alkenyl
  • a 1 is (CH 2 ) 1-2
  • a 2 is O
  • a 3 is (CH 2 ) 1-5 , wherein A 3 is not CH 2 if X is N, X is N, CH, or C-CH 3
  • a 6 is O, S, NH, NCH 3 or (CH 2 ) 0-2
  • a 7 is (CH 2 ) 0-6 , wherein if A 6 is O, S, NH, NCH 3 , A 7 is (CH 2 ) 2-4 , each W is independently CH or N;
  • each R 2 is independently a C 7 -C 11 alkyl or a C
  • the ionizable cationic lipid of embodiment 1, wherein in each tail group, both R 2 groups are O and both R 3 groups are C O.
  • Embodiment 8. The ionizable cationic lipid of embodiment 6, wherein each W is N.
  • Embodiment 9. The ionizable cationic lipid of any one of embodiments 1, 3, 5, or 6 wherein both R 3 groups at a beta position of a W are C O and that W is N.
  • the ionizable cationic lipid of any one of embodiments 1-12, wherein A 1 is CH 2 , A 3 is (CH 2 ) 2-5 , X is N, A 4 is C O, A 5 is O, S, NH, or NCH 3 , A 6 is CH 2 , A 7 is (CH 2 ) 0-6, wherein if A 6 is O, NH, NCH 3 , A 7 is (CH 2 ) 2-4 .
  • Embodiment 14 The ionizable cationic lipid of embodiment 13, wherein A 5 is O.
  • the ionizable cationic lipid of any one of embodiments 1-12, wherein A 1 is CH 2 , A 3 is (CH 2 ) 1-4 , X is CH, A 4 is CH 2 , NH, NCH 3 , O, A 5 is C O, A 6 is O, NH, NCH 3 , or CH 2 , A 7 is (CH) 2-6 .
  • Embodiment 16. The ionizable cationic lipid of embodiment 15, wherein A 4 is NH.
  • Embodiment 17 The ionizable cationic lipid of embodiment 15, wherein A 4 is NH and A 6 is O.
  • Embodiment 18. The ionizable cationic lipid of embodiment 15, wherein A 4 is NH and A 6 is CH 2 .
  • Embodiment 19 The ionizable cationic lipid of embodiment 15, wherein A 4 is CH 2 .
  • Embodiment 20 The ionizable cationic lipid of embodiment 15, wherein A 4 is CH 2 and A 6 is O.
  • Embodiment 21 The ionizable cationic lipid of embodiment 15, wherein A 4 is O.
  • Embodiment 22 The ionizable cationic lipid of embodiment 15, wherein A 4 is O and A 6 is CH 2 .
  • Embodiment 23 The ionizable cationic lipid of embodiment 15, wherein A 4 is NCH 3 .
  • Embodiment 24 The ionizable cationic lipid of embodiment 15, wherein A 4 is NCH 3 and A 6 is CH 2 .
  • Embodiment 26 The ionizable cationic lipid of embodiment 25, wherein A 5 is O.
  • Embodiment 27 The ionizable cationic lipid of any one of embodiments 1-25, wherein O the number of contiguous atoms present in at least one span: is in the range of 10 to 17.
  • Embodiment 30. The ionizable cationic lipid of any one of embodiments 1-12, wherein A 1 Embodiment 31.
  • Embodiment 32. The ionizable cationic lipid of embodiment 30, wherein A 4 is CH 2 .
  • Embodiment 33 The ionizable cationic lipid of embodiment 30, wherein A 4 is O.
  • Embodiment 34 The ionizable cationic lipid of embodiment 30, wherein A 4 is NCH 3 .
  • Embodiment 35 The ionizable cationic lipid of any one of embodiments 1-12, wherein A 1 absent, A 6 is (CH 2 ) 0 , A 7 is (CH 2 ) 0 , and Y Embodiment 36.
  • Embodiment 37 The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 39.
  • Embodiment 40 The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond.
  • Embodiment 41 The ionizable cationic lipid of any one of embodiments 1 to 36, wherein OCH 3 Z N Y is and Z is a bond.
  • Embodiment 42 The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond.
  • Embodiment 43 The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond.
  • Embodiment 52. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 54.
  • Embodiment 56. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 56.
  • Embodiment 58. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 60.
  • Embodiment 65 The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond.
  • Embodiment 63 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is independently C7-C 10 alkyl or C 7 -C 9 alkyl.
  • Embodiment 64 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is independently a linear C 7 -C 11 alkyl, e.g., a linear C 7 -C 10 alkyl, or a linear C 7 -C 9 alkyl.
  • Embodiment 65 Embodiment 65.
  • each R 1 is independently (CH 2 ) 6-8 CH 3 .
  • Embodiment 66 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is (CH 2 ) 7 CH 3 .
  • Embodiment 67 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is independently a linear C 7 -C 11 alkenyl, e.g., a linear C 7 -C 10 alkenyl, or a linear C 7 -C 9 alkenyl.
  • Embodiment 68 Embodiment 68.
  • each R 1 is a linear C 8 alkenyl.
  • Embodiment 69 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is independently a branched C 7 -C 11 alkyl, e.g., C 7 -C 10 alkyl, or C 7 -C 9 alkyl.
  • Embodiment 70 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is a branched C 8 alkyl.
  • Embodiment 71 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is a branched C 8 alkyl.
  • each R 1 is independently a branched C 7 -C 11 alkenyl, e.g., C 7 -C 10 alkenyl, or C 7 -C 9 alkenyl.
  • Embodiment 72 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is a branched C 8 alkenyl.
  • Embodiment 73 The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R 1 is a branched C 8 alkenyl.
  • Embodiment 74. The ionizable cationic lipid of any one of embodiments 1 to 73, wherein each R 1 is the same.
  • Embodiment 77 The ionizable cationic lipid of embodiment 76, wherein A 5 is O.
  • Embodiment 80. The ionizable cationic lipid of embodiment 78, wherein A 4 is NH and A 6 is O.
  • Embodiment 81 The ionizable cationic lipid of embodiment 78, wherein A 4 is NH and A 6 is CH 2 .
  • Embodiment 82 The ionizable cationic lipid of embodiment 78, wherein A 4 is CH 2 .
  • Embodiment 83 The ionizable cationic lipid of embodiment 78, wherein A 4 is CH 2 and A 6 is O.
  • Embodiment 84. The ionizable cationic lipid of embodiment 78, wherein A 4 is O.
  • Embodiment 85 The ionizable cationic lipid of embodiment 78, wherein A 4 is O and A 6 is CH 2 .
  • Embodiment 86 The ionizable cationic lipid of embodiment 78, wherein A 4 is O and A 6 is CH 2 .
  • Embodiment 87. The ionizable cationic lipid of embodiment 78, wherein A 4 is NCH 3 and A 6 is CH 2 .
  • Embodiment 90. The ionizable cationic lipid of any one of embodiments 75-89, wherein O A 3 4 6 the number of contiguous atoms present in at least one span: A1 A2 A X A 5 A A 7 i s in the range of 10 to 17.
  • Embodiment 93 The ionizable cationic lipid of embodiment 75, wherein A 1 is CH 2 , A 3 is and Y is Embodiment 94.
  • Embodiment 95 The ionizable cationic lipid of embodiment 93, wherein A 4 is CH 2 .
  • Embodiment 96 The ionizable cationic lipid of embodiment 93, wherein A 4 is O.
  • Embodiment 97 The ionizable cationic lipid of embodiment 93, wherein A 4 is NCH 3 .
  • Embodiment 98 The ionizable cationic lipid of embodiment 75, wherein A 1 is (CH 2 ) 2 , A 3 absent, A 6 is (CH 2 ) 0 , A 7 is (CH 2 ) 0 , and Y is Embodiment 99.
  • Embodiment 100 Embodiment 100.
  • Embodiment 101 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond.
  • Embodiment 101 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein ( CH2)0-1 CH 3 Z N Y is (CH2)0-1 CH 3 and Z is a bond.
  • Embodiment 102 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond.
  • Embodiment 103 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond.
  • Embodiment 104 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein OCH 3 Z N Y is and Z is a bond.
  • Embodiment 105 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 107.
  • Embodiment 112. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 114.
  • Embodiment 119. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 121.
  • Embodiment 126 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond.
  • Embodiment 126 The ionizable cationic lipid of any one of embodiments 75 to 125, wherein W is CH.
  • Embodiment 127 The ionizable cationic lipid of any one of embodiments 75 to 125, wherein W is N.
  • Embodiment 128 The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R 1 is independently C 7 -C 10 alkyl or C 7 -C 9 alkyl.
  • Embodiment 129 The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond.
  • Embodiment 126 The ionizable cationic lipid of any one of embodiments 75 to 125, wherein W is CH.
  • Embodiment 127 The ionizable cationic lipid of any one of embodiments
  • each R 1 is independently a linear C 7 -C 11 alkyl, e.g., a linear C 7 -C 10 alkyl, or a linear C 7 -C 9 alkyl.
  • Embodiment 130 The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R 1 is independently (CH 2 ) 6-8 CH 3 .
  • Embodiment 131 The ionizable cationic lipid of any one of embodiments 75 to 127, wherein R 1 is (CH 2 ) 7 CH 3 .
  • Embodiment 132 is
  • each R 1 is independently a linear C 7 -C 11 alkenyl, e.g., a linear C 7 -C 10 alkenyl, or a linear C 7 -C 9 alkenyl.
  • Embodiment 133 The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R 1 is a linear C 8 alkenyl.
  • Embodiment 134 The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R 1 is a linear C 8 alkenyl.
  • each R 1 is independently a branched C 7 -C 11 alkyl, e.g., C 7 -C 10 alkyl, or C 7 -C 9 alkyl.
  • Embodiment 135. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R 1 is a branched C 8 alkyl.
  • each R 1 is independently a branched C 7 -C 11 alkenyl, e.g., C 7 -C 10 alkenyl, or C 7 -C 9 alkenyl.
  • Embodiment 137 The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R 1 is a branched C 8 alkenyl.
  • Embodiment 138 The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R 1 is a branched C 8 alkenyl.
  • Embodiment 140. The ionizable cationic lipid of embodiment 75 having the structure CICL-250: CICL-250 .
  • Embodiment 142 The ionizable cationic lipid of embodiment 75 having the structure CICL-292: . Embodiment 143.
  • the ionizable cationic lipid of embodiment 75 having the structure CICL-297 .
  • Embodiment 1 having a structure as shown in Table 2.
  • Embodiment 155 The ionizable cationic lipid of embodiment 1 having a structure as shown in Table 3.
  • Embodiment 156 The ionizable cationic lipid of embodiment 1 having a structure as shown in Table 4.
  • Embodiment 157 The ionizable cationic lipid of any one of embodiments 1 to 156 having a -pKa (calculated pKa) in the range of from about 6, 7, or 8 to about 9, 10, or 11.
  • Embodiment 158 The ionizable cationic lipid of any one of embodiments 1 to 156 having a -pKa (calculated pKa) in the range of from about 6, 7, or 8 to about 9, 10, or 11.
  • the ionizable cationic lipid of any one of embodiments 1 to 160 having a cLogD ranging from 9 to 18, for example, ranging from 10 to 18, or 10 to 16, to 10 to 14, or 11 to 18, or 11 to 15, or 11 to 14, or 12 to 14.
  • the ionizable cationic lipid of any one of embodiments 1 to 160 having a cLogD is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18.
  • the ionizable cationic lipid of any one of embodiments 1 to 160 having a cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4.
  • the ionizable cationic lipid of any one of embodiments 1 to 160 having a c-pKa ranging from about 8 to about 11 or from 8 to 11 and a cLogD ranging from about 9 to about 18 or from 9 to 18.
  • the ionizable cationic lipid of any one of embodiments 1 to 160 having a c-pKa ranging from about 8.4 to about 8.7 or from 8.4 to 8.7 and cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4.
  • Embodiment 167. A lipid nanoparticle (LNP), comprising at least one ionizable cationic lipid of any one of embodiments 1-166.
  • a targeted lipid nanoparticle (tLNP) comprising a lipid nanoparticle of embodiment 167 and a functionalized PEG-lipid, wherein the functionalized PEG-lipid has been conjugated with a binding moiety.
  • the LNP or tLNP of embodiment 169 wherein the phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof.
  • DOPE dioleoylphosphatidyl ethanolamine
  • DMPC dimyristoylphosphatidyl choline
  • DSPC distearoylphosphatidylcholine
  • DMPG dimyristoylphosphatidyl glycerol
  • DPPC dipalmitoyl phosphatidylcholine
  • DAPC 1,2-diarachidoyl-sn-gly
  • Embodiment 172 The LNP or tLNP of any one of embodiments 169-170, wherein the co- lipid comprises cholesterol hemisuccinate (CHEMS) or a quaternary ammonium head group containing lipid.
  • the LNP or tLNP of embodiment 172 wherein the quaternary ammonium head group containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3 ⁇ -(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium propane
  • DOTMA N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
  • DC-Chol 3 ⁇ -(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol
  • the LNP of any one of embodiments 169-174 or the tLNP of any one of embodiments 168-174, wherein the unfunctionalized PEG-lipid and/or the functionalized PEG-lipid comprises fatty acids with a fatty acid chain length of C 14 -C 18 .
  • Embodiment 176 Embodiment 176.
  • Embodiment 177 The LNP or tLNP of any one of embodiments 169-176, comprising a phospholipid in an amount in the range from 7 to 30 mol%.
  • Embodiment 178. The LNP or tLNP of any one of embodiments 169-177, comprising a sterol in an amount in the range from 20 to 45 mol%.
  • Embodiment 179. The LNP or tLNP of any one of embodiments 169-178, comprising at least one co-lipid in an amount in the range from 1 to 30 mol%.
  • Embodiment 180 The LNP or tLNP of any one of embodiments 169-176, comprising a phospholipid in an amount in the range from 7 to 30 mol%.
  • Embodiment 178. The LNP or tLNP of any one of embodiments 169-177, comprising a sterol in an amount in the range from 20 to 45 mol%.
  • Embodiment 179. The LNP or
  • the LNP or tLNP of any one of embodiments 169-179 comprising at least one unfunctionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%.
  • Embodiment 181. The LNP or tLNP of any one of embodiments 169-180, comprising at least one functionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%.
  • Embodiment 182. The tLNP of any one of embodiments 169-181, wherein the binding moiety comprises an antigen, a ligand-binding domain of a receptor, a receptor ligand, an antibody, or an antigen binding domain of an antibody.
  • a method of delivering a biologically active payload (e.g., one or more species of nucleic acid molecule) into a cell comprising contacting the cell with the LNP or tLNP any one of embodiments 183-192.
  • Embodiment 195 The lipid of embodiment 194, wherein R 1 , R 2 , R 3 , and W are as otherwise described herein.
  • Embodiment 196. The lipid of embodiment 194 or embodiment 195, wherein R 4 is H.
  • Embodiment 197 The lipid of embodiment 194 or embodiment 195, wherein R 4 is a protecting group (e.g., t-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), or a trimethylsilylethoxycarbonyl moiety.
  • R 4 is a protecting group (e.g., t-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), or a trimethylsilylethoxycarbonyl moiety.
  • the lipid of any one of embodiments 194-197 wherein when A 1 and A 3 are CH 2 , then X is CH, A 4 is CH 2 , NH, NCH 3 , or O.
  • Embodiment 199 The lipid of any one of embodiments 194-197, wherein when A 1 and A 3 are CH 2 , then X is CH, and A 4 is NH 2 .
  • Embodiment 200 The lipid of any one of embodiments 194-197, wherein when A 1 is CH 2 and A 3 is CH 2 or CH 2 CH 2 , and X is CH, then A 4 is NH, NCH 3 , or O.
  • Embodiment 201 The lipid of any one of embodiments 194-197, wherein when A 1 is CH 2 and A 3 is CH 2 or CH 2 CH 2 , and X is CH, then A 4 is NH, NCH 3 , or O.
  • the lipid of any one of embodiments 194-197 wherein when A 1 is CH 2 , A 3 is CH 2 CH 2 , X is CH, and A 4 is O.
  • Embodiment 202 The lipid of any one of embodiments 194-197, wherein when A 1 is CH 2 , A 3 is CH 2 CH 2 , X is C-CH 3 , and A 4 is NH.
  • Embodiment 203 The lipid of any one of embodiments 194-197, wherein when A 1 is CH 2 , A 3 is CH 2 CH 2 , X is C-CH 3 , and A 4 is NCH 3 .
  • Embodiment 204 The lipid of any one of embodiments 194-197, wherein when A 1 is CH 2 , A 3 is CH 2 CH 2 , X is C-CH 3 , and A 4 is NCH 3 .
  • Embodiment 204 The lipid of embodiment 204, wherein R 1 , R 2 , R 3 , and W are as otherwise described herein.
  • Embodiment 206 The lipid of embodiment 204 or embodiment 205, wherein R 5 is OH.
  • Embodiment 207 The lipid of embodiment 204 or embodiment 205, wherein R 5 is .
  • Embodiment 208. The lipid of any one of embodiments 204-207, wherein when A 1 and A 3 are CH 2 , then X is CH, A 4 is CH 2 , NH, NCH 3 , or O.
  • Embodiment 209 The lipid of any one of embodiments 204-207, wherein A 1 and A 3 are Embodiment 210.
  • the lipid of embodiment 211, wherein R 1 , R 2 , R 3 , and W are as otherwise described herein.
  • Embodiment 213. The lipid of embodiment 211 or embodiment 212, wherein R 6 is OH.
  • Embodiment 215. The lipid of any one of embodiments 211-214, wherein A 1 is CH 2 or CH 2 CH 2 , A 3 is CH 2 or CH 2 CH 2 , and X is C-CH 3 .
  • Embodiment 216. The lipid of any one of embodiments 211-214, wherein A 1 is CH 2 , A 3 is CH 2 CH 2 , X is C-CH 3 , and R 6 is OH.
  • Embodiment 217 The lipid of any one of embodiments 211-214, wherein A 1 is CH 2 or CH 2 CH 2 , A 3 is CH 2 or CH 2 CH 2 , and X is N.
  • Embodiment 218 The lipid of any one of embodiments 211-214, wherein A 1 is CH 2 , A 4 is Embodiment 219.
  • a lipid having the structure of formula I4-1, wherein each R 1 is independently a C 7 -C 11 alkyl or a C 7 -C 11 alkenyl; each W is independently CH or N; A 1 is (CH 2 ) 1-2 , A 2 is O, A 3 is (CH 2 ) 1-5 , wherein A 3 is not CH 2 if X is N, X is N, CH, or C-CH 3 , A 4 is CH 2 , C O, NH, NCH 3 , or O, and R 4 is H or a protecting group.
  • Embodiment 220 The lipid of embodiment 219, wherein R 1 and W are as otherwise described herein.
  • Embodiment 222 The lipid of embodiment 219 or embodiment 220, wherein R 4 is H.
  • Embodiment 222 The lipid of embodiment 219 or embodiment 220, wherein R 4 is a protecting group (e.g., t-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), or a trimethylsilylethoxycarbonyl moiety.
  • Embodiment 223. The lipid of any one of embodiments 219-222, wherein when A 1 and A 3 are CH 2 , then X is CH, A 4 is CH 2 , NH, NCH 3 , or O.
  • Embodiment 224 Embodiment 224.
  • Embodiment 227 The lipid of any one of embodiments 219-222, wherein when A 1 and A 3 are CH 2 , then X is CH, and A 4 is NH 2 .
  • Embodiment 225 The lipid of any one of embodiments 219-222, wherein when A 1 is CH 2 and A 3 is CH 2 or CH 2 CH 2 , and X is CH, then A 4 is NH, NCH 3 , or O.
  • Embodiment 226 The lipid of any one of embodiments 219-222, wherein when A 1 is CH 2 , A 3 is CH 2 CH 2 , X is CH, and A 4 is O.
  • Embodiment 227 The lipid of any one of embodiments 219-222, wherein when A 1 is CH 2 , A 3 is CH 2 CH 2 , X is CH, and A 4 is O.
  • the lipid of embodiment 229, wherein R 1 and W are as otherwise described herein.
  • Embodiment 236 The lipid of embodiment 229 or embodiment 230, wherein R 5 is .
  • Embodiment 233 The lipid of any one of embodiments 229-232, wherein when A 1 and A 3 are CH 2 , then X is CH, A 4 is CH 2 , NH, NCH 3 , or O.
  • the lipid of any one of embodiments 229-232, wherein A 1 and A 3 are Embodiment 235.
  • Embodiment 236 The lipid of any one of embodiments 229-232, wherein A 1 and A 3 are CH 2 , X is CH, A 5 is CH 2 , and R 5 is OH.
  • the lipid of embodiment 236, wherein R 1 and W are as otherwise described herein.
  • the lipid of embodiment 236 or embodiment 237, wherein R 6 is OH.
  • Embodiment 240 The lipid of any one of embodiments 236-239, wherein A 1 is CH 2 or CH 2 CH 2 , A 3 is CH 2 or CH 2 CH 2 , and X is C-CH 3 .
  • Embodiment 241. The lipid of any one of embodiments 236-239, wherein A 1 is CH 2 , A 3 is CH 2 CH 2 , X is C-CH 3 , and R 6 is OH.
  • Embodiment 243 The lipid of any one of embodiments 236-239, wherein A 1 is CH 2 or CH 2 CH 2 , A 3 is CH 2 or CH 2 CH 2 , and X is N.
  • a method of treating a disease or disorder comprising administration of a tLNP of any one of embodiments 183-190 to a subject in need thereof.
  • Embodiment 245. The method of embodiment 244, wherein the disease or disorder is an autoimmune disease.
  • Embodiment 246. The method of embodiment 244, wherein the autoimmune disease is a T cell-mediated autoimmunity or a B-cell mediated autoimmunity.
  • Embodiment 247 The method of embodiment 244, wherein the disease or disorder is a rejection of an allogeneic organ or tissue graft.
  • the method of embodiment 244, wherein the disease or disorder is cancer.
  • Embodiment 249. The method of embodiment 244, wherein the disease or disorder is a fibrotic disease or disorder.
  • the method of embodiment 244, wherein the disease or disorder is a graft versus host disease (GVHD).
  • Embodiment 251. A method of treating a disease or disorder comprising administration of a tLNP of any one of embodiments 183-188 or 191-192 to a subject in need thereof.
  • Embodiment 252. The method of embodiment 251, wherein the disease or disorder is a genetic disease or disorder.
  • Z N [00451] Throughout this disclosure embodiments are described in which Y is , (wherein Z is a bond).

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biomedical Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Nanotechnology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Optics & Photonics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Ionizable cationic lipids, methods for synthesizing the same, intermediates useful in synthesis of the ionizable cationic lipids, and methods of synthesizing the intermediates are disclosed. The ionizable cationic lipids are useful as a component of lipid nanoparticles (LNP), which in turn can be used for delivering nucleic acids into cells in vivo or ex vivo. LNP compositions are also disclosed, including LNP comprising a functionalized lipid to enable conjugation of a binding moiety, and targeted LNP (tLNP), that is an LNP in which a binding moiety has been conjugated to the functionalized lipid and can serve as a targeting moiety to direct the tLNP to a desired tissue or cell type.

Description

IONIZABLE CATIONIC LIPIDS AND LIPID NANOPARTICLES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority U.S. provisional application number 63/632,940, filed April 11, 2024, U.S. provisional application number 63/654,704, filed May 31, 2024, U.S. provisional application number 63/654,930, filed May 31, 2024, U.S. provisional application number 63/654,928, filed May 31, 2024, U.S. provisional application number 63/708,461, filed October 17, 2024, and U.S. provisional application number 63/708,529, filed October 17, 2024; the disclosures of which are expressly incorporated by reference herein. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [0002] The instant application contains a Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing was created on March 12, 2025, is named “CTX-020PR3_24-0470-US-PRO3_Sequence Listing.xml”, and is 3,692 bytes in size. BACKGROUND [0003] Lipid formulations have been used in the laboratory for delivering nucleic acids into cells. Early formulations that were based on the cationic lipid 1,2-dioleoyl-3- trimethylammonium propane (DOTAP) and the ionizable, fusogenic lipid dioleoylphosphatidyl ethanolamine (DOPE) had a large particle size and were problematic when used in vivo, exhibiting too rapid clearance, tropism for the lung, and toxicity. Lipid nanoparticles (LNPs) comprising ionizable cationic lipids have been developed to address these issues to the extent that RNA-based products, such as the siRNA ONPATTRO® and two mRNA-based SARS-CoV-2 vaccines have received regulatory approval and entered the marketplace. [0004] However, there is limited ability to control which tissues or cells take up the LNP once administered. LNP administered intravenously are taken up primarily in the liver, lung, or spleen depending to a significant degree on net charge and particle size. It is possible to direct >90% of LNP to the liver by a combination of formulation and intravenous administration, for example. Intramuscular administration can provide a clinically useful level of local delivery and expression. LNP can be redirected to other tissues or cell types by conjugating to the LNP a binding moiety with specificity for the target tissue or cell type, for example, conjugating an antibody to an LNP (see, e.g., Endsley and Ho, J. Acquir. Immune Defic. Syndr.61:417, 2012; Ramishetti et al., ACS Nano 9:6706, 2015; Veiga et al., Nat. Comms.9:4493, 2018; US Patent No.10,920,246). Nonetheless, avoiding uptake by the liver remains a challenge. Moreover, with current systems only a minor portion of the encapsulated nucleic acid is successfully delivered to the cells of interest and into the cytoplasm. Current formulations may release only 2-5% of the administered RNA into the cytoplasm (see, for example, Gilleron et al., 2013, Nat. Biotechnol.31:638-646 and Munson et al., 2021, Commun. Biol.4:211-224). There are remaining issues of off-target delivery, poor efficiency of release of nucleic acid into the cytoplasm, and toxicity associated with accumulation of the component lipids. [0005] Thus, there exists a need to address issues of off-target delivery, poor efficiency of release of nucleic acid into the cytoplasm, and toxicity associated with accumulation of the component lipids. SUMMARY [0006] This disclosure fulfills the needs for addressing issues of off-target delivery, poor efficiency of release of therapeutic agents and provides further related advantages. [0007] In certain aspects, this disclosure provides ionizable cationic lipids having a structure of formula M5 set forth herein. [0008] In certain aspects, this disclosure provides ionizable cationic lipids having a structure of formula M4 set forth herein. [0009] In some embodiments, this disclosure provides ionizable cationic lipids CICL-250, CICL-291, CICL-292, CICL-293, CICL-294, CICL-295, CICL-296, CICL-297, CICL-298, CICL-299, CILC-300, CICL-301, CICL-302, CICL-303, CICL-309, CICL-310, and CICL-311 set forth herein. [0010] In some embodiments, this disclosure provides ionizable cationic lipids CICL- 250-61, CICL-250-62, CICL-250-63, CICL-250-64, CICL-250-65, CICL-250-66, CICL-250- 67, CICL-250-68, CICL-250-69, CICL-250-70, CICL-250-71, CICL-250-72, CICL-250-73, CICL-250-74, CICL-250-75, CICL-250-76, CICL-250-77, CICL-291-61, CICL-291-62, CICl- 291-63, CICL-291-65, CICL-291-67, CICL-291-68, CICL-291-72, CICL-291-75, CICL-292- 61, CICL-292-62, CICL-292-63, CICL-292-65, CICL-292-67, CICL-292-68, CICL-292-72, CICL-292-75, CICL-296-61, CICL-296-62, CICL-296-63, CICL-296-65, CICL-296-67, CICL- 296-68, CICL-296-72, CICL-296-75, CICL-297-61, CICL-297-62, CICL-297-63, CICL-297- 65, CICL-297-67, CICL-297-68, CICL-297-72, CICL-297-75, CICL-298-61, CICL-298-62, CICL-298-63, CICL-298-65, CICL-298-67, CICL-298-68, CICL-298-72, CICL-298-75, CICL- 299-61, CICL-299-62, CICL-299-63, CICL-299-65, CICL-299-67, CICL-299-68, CICL-299- 72, CICL-299-75, CICL-303-61, CICL-303-62, CICL-303-63, CICL-303-65, CICL-303-67, CICL-303-68, CICL-303-72, and CICL-303-75 set forth herein. [0011] In certain aspects, this disclosure provides methods of synthesizing ionizable cationic lipids as described herein, e.g. CICL-250, CICL-291, CICL-292, CICL-293, CICL- 294, CICL-295, CICL-296, CICL-297, CICL-298, CICL-299, CILC-300, CICL-301, CICL-302, CICL-303, CICL-309, CICL-310, and CICL-311 set forth herein. [0012] In certain aspects, this disclosure provides methods of synthesizing ionizable cationic lipids as described herein, e.g., CICL-250-61, CICL-250-62, CICL-250-63, CICL- 250-64, CICL-250-65, CICL-250-66, CICL-250-67, CICL-250-68, CICL-250-69, CICL-250- 70, CICL-250-71, CICL-250-72, CICL-250-73, CICL-250-74, CICL-250-75, CICL-250-76, CICL-250-77, CICL-291-61, CICL-291-62, CICl-291-63, CICL-291-65, CICL-291-67, CICL- 291-68, CICL-291-72, CICL-291-75, CICL-292-61, CICL-292-62, CICL-292-63, CICL-292- 65, CICL-292-67, CICL-292-68, CICL-292-72, CICL-292-75, CICL-296-61, CICL-296-62, CICL-296-63, CICL-296-65, CICL-296-67, CICL-296-68, CICL-296-72, CICL-296-75, CICL- 297-61, CICL-297-62, CICL-297-63, CICL-297-65, CICL-297-67, CICL-297-68, CICL-297- 72, CICL-297-75, CICL-298-61, CICL-298-62, CICL-298-63, CICL-298-65, CICL-298-67, CICL-298-68, CICL-298-72, CICL-298-75, CICL-299-61, CICL-299-62, CICL-299-63, CICL- 299-65, CICL-299-67, CICL-299-68, CICL-299-72, CICL-299-75, CICL-303-61, CICL-303- 62, CICL-303-63, CICL-303-65, CICL-303-67, CICL-303-68, CICL-303-72, and CICL-303-75. [0013] In certain aspects, this disclosure provides intermediates and methods of synthesizing such intermediates useful in the synthesis of the ionizable cationic lipids as described herein. [0014] In certain aspects, this disclosure provides lipid nanoparticles (LNPs) and targeted lipid nanoparticles (tLNPs) incorporating the ionizable cationic lipids disclosed herein. [0015] In certain aspects, this disclosure provides methods for preparing LNPs and tLNPs as described herein. [0016] In certain embodiments, this disclosure provides methods of delivering a biologically active payload (e.g., nucleic acid molecules encoding a therapeutic agent) into a cell comprising contacting the cell with an LNP or tLNP of this disclosure. [0017] These and other features, objects, and advantages of this disclosure will become better understood from the description that follows. In the description, reference is made to the accompanying drawings, which form a part hereof and in which there is shown by way of illustration, not limitation, embodiments of this disclosure. The description of preferred embodiments is not intended to limit this disclosure from covering all modifications, equivalents, and alternatives. Reference should therefore be made to the claims recited herein for interpreting the scope of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS [0018] The disclosure is better understood and features, aspects, and advantages other than those set forth above will become apparent when consideration is given to the following description of the drawings. [0019] Figures 1A-1C depict the transfection rate (percentage of cells expressing the mRNA) versus expression level (as molecules of equivalent soluble fluorochrome (MESF)) for two tLNP compositions with CICL-250 or CICL-1, as indicated. The full lipid compositions are described in Table 5. Results in spleen T cells (Figure 1A), CD45- liver cells (Figure 1B) and liver Kupffer cells (CD45+/CD11+ liver cells; Figure 1C) from C57BL/6 mice administered the tLNPs are shown. The binding moiety of the tLNPs was an anti-CD5 antibody and the payload was an mRNA encoding mCherry (SEQ ID NO: 2). DETAILED DESCRIPTION [0020] This disclosure provides ionizable cationic lipids, referred to as ionizable cationic lipids throughout, methods for synthesizing them, as well as intermediates useful in synthesis of these lipids and methods of synthesizing the intermediates. This disclosure further provides ionizable cationic lipids as components of lipid nanoparticles (LNPs) that can be used for delivering a biologically active payload (e.g., nucleic acid molecules encoding a therapeutic agent) into cells in vivo or ex vivo. LNP compositions are also disclosed herein, including LNPs comprising a functionalized PEG-lipid to enable conjugation of a binding moiety to generate targeted LNPs (tLNPs); that is, LNPs containing a binding moiety that directs the tLNP to a desired tissue or cell type (e.g., immune cells such as T cells or stem cells such as hematopoietic stem cells (HSCs)). Also disclosed herein are methods of delivering a nucleic acid molecule into a cell comprising contacting the cell with an LNP or tLNP of this disclosure. The LNP and tLNP of this disclosure can be used for in vivo, ex vivo, or extracorporeal transfection. Also disclosed herein are methods for preparing LNPs and tLNPs comprising the ionizable cationic lipids as described herein. [0021] Prior to setting forth this disclosure in more detail, it may be helpful to provide abbreviations of certain terms used herein. Additional abbreviations are set forth throughout this disclosure. ABBREVIATIONS [0022] Abbreviations used herein include: [0023] While this disclosure is capable of being embodied in various forms, the description below of several embodiments is made with the understanding that the disclosure is to be considered as an exemplification of the innovations disclosed herein and is not intended to limit the disclosure to the specific embodiments illustrated. [0024] Headings are provided for convenience only and are not to be construed to limit the embodiments in any manner. Embodiments illustrated under any heading can be combined with embodiments illustrated under any other heading. [0025] To the extent any materials incorporated herein by reference conflict with this disclosure, this disclosure controls. DEFINITIONS [0026] Prior to setting forth this disclosure in more detail, definitions of certain terms to be used herein are provided. Additional definitions are set forth throughout this disclosure. [0027] As used in the specification and claims, the singular form “a,” “an,” and “the” includes plural references unless the context clearly dictates otherwise. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. [0028] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. [0029] The term “about” as used herein in the context of a number refers to a range centered on that number and spanning 10% less than that number and 10% more than that number. The term “about” used in the context of a range refers to an extended range spanning 10% less than that of the lowest number listed in the range and 10% more than the greatest number listed in the range. For any value modified by the word “about,” it should be understood to also disclose an embodiment wherein the value is not so modified. [0030] Throughout this disclosure, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range of this disclosure relating to any physical feature, such as polymer subunits, size, or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. Throughout this disclosure, numerical ranges are inclusive of their recited endpoints, unless specifically stated otherwise. [0031] Unless the context requires otherwise, throughout this specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used herein, the terms “include” and “comprise” are used synonymously. [0032] The phrase “at least one of” when followed by a list of items or elements refers to an open-ended set of one or more of the elements in the list, which can, but does not necessarily, include more than one of the elements. [0033] “Derivative,” as used herein, refers to a chemically or biologically modified version of a compound that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. Generally, a “derivative” differs from an “analogue” in that a parent compound can be the starting material to generate a “derivative,” whereas the parent compound can not necessarily used as the starting material to generate an “analogue.” A derivative can have different chemical or physical properties than the parent compound. For example, a derivative can be more hydrophilic or hydrophobic, or it can have altered reactivity as compared to the parent compound. Although a derivative can be obtained by physical (for example, biological or chemical) modification of the parent compound, a derivative can also be conceptually derived, for example, as when a protein sequence is designed based on one or more known sequences, an encoding nucleic acid is constructed, and the derived protein obtained by expression of the encoding nucleic acid. [0034] As used herein “extracorporeal” is used with reference to cells, such as peripheral blood or bone marrow cells, harvested or extracted from the body and the manipulation or modification of those cells prior to their intended return (reinfusion). Manipulation and modification of cells generally relates to cell separation and washing procedures and exposure to activation agents (e.g., biological response modifiers (BRMs)) and transfection agents (e.g., LNPs, tLNPs), over a time interval of several hours, for example, less than 6 hours, less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour; and in space to a single institution. Extracorporeal is used in contradistinction to ex vivo which, as used herein, includes more extensive manipulation including extended periods of cell culture and expansion, and/or refrigerated or cryogenic storage or shipment, over several days or longer. [0035] As used herein “expansion” refers to proliferation of cells increasing their number. Activating agents can be used to stimulate proliferation (among other metabolic changes) but can also result in activation-induced death upon initial exposure so that there is no immediate expansion. For T cells treated in vitro with activating agents such as IL-2 or CD3/CD28 activators, doubling time can be about 24 hours (which is fairly typical of mammalian cells in vitro generally); in vivo doubling time can be substantially shorter, depending on the presence and type of stimulation. Accordingly, during a limited time of extracorporeal manipulation, even when activating agents are used, such protocols will be effectively expansion-less. [0036] As used herein an “exogenous protein” refers to a synthetic, recombinant, or other peptide or protein that is not produced by a wild-type cell of that type or is expressed at a lower level in a wild-type cell than in a cell containing the exogenous polypeptide, or that is administered to a subject rather than being produced inside the subject’s body. In some embodiments, an exogenous peptide is a peptide or protein encoded by a nucleic acid that was introduced into the cell, which nucleic acid is optionally not retained by the cell. As used herein “peptide” refers to a chain of amino acids less than 50 amino acids in length, while “protein” and “polypeptide refer to a chain of amino acids at least 50 amino acids in length. [0037] As used herein “transfection” or “transfecting” refers to the introduction of nucleic acids into cells by non-viral methods. Transfection can be mediated by calcium phosphate, cationic polymers, magnetic beads, electroporation, and lipid-based reagents. In preferred embodiments disclosed herein transfection is mediated by solid lipid nanoparticles (LNP) including targeted LNP (tLNP) (which can also be used to deliver non-nucleic acid payloads into cells). The term transfection is used in distinction to transduction – transfer of genetic material from cell to cell or virus to cell – and transformation – the uptake of extracellular genetic material by the natural processes of a cell. As used herein, phrases such as “delivering a nucleic acid into a cell” are synonymous with transfection. [0038] “Reprogramming,” as used herein with respect to cells, refers to changing the functionality of a cell. For example, reprogramming an immune cell can result in a change in antigenic specificity by causing expression of an exogenous T cell receptor (TCR), a chimeric antigen receptor (CAR), or an immune cell engager (“reprogramming agents”). Generally, T lymphocytes and natural killer (NK) cells can be reprogrammed with a TCR, a CAR, or an immune cell engager, while a CAR or an immune cell engager are typically used in reprogramming monocytes. As used herein with respect to stem cells, for example hematopoietic stem cells (HSC) or mesenchymal stem cells (MSC), “reprogramming” refers to correction or amelioration of a genetic defect (for example, a hemoglobinopathy) so that the modified or corrected gene and gene product are the reprogramming agents. Reprogramming can be transient or durable depending on the nature of the engineering agent. [0039] “Engineering agent,” as used herein, refers to agents that confer expression of a reprogramming agent by a cell, such as an immune cell, particularly a non-B lymphocyte or monocyte. Engineering agents can include nucleic acid molecules, including mRNA, that encode a reprogramming agent. Engineering agents can also include nucleic acid molecules that are or encode components of gene editing systems such as RNA-guided nucleases, guide RNA, and nucleic acid templates for knocking-in a reprogramming agent or knocking- out an endogenous antigen receptor. Gene editing systems comprise base-editors, prime- editors or gene-writers. RNA-guided nucleases include CRISPR nucleases such as Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, and CasX. For transient expression of a reprogramming agent, such as a CAR, an mRNA encoding the reprogramming agent can be used as the engineering agent. For durable expression of the reprogramming agent, such as an exogenous, modified, or corrected gene (and its gene product), the engineering agent can comprise mRNA-encoded RNA-directed nucleases, guide RNAs, nucleic acid templates and other components of gene/genome editing systems. [0040] Examples of gene editing components that are encoded by a nucleic acid molecule include an mRNA encoding an RNA-guided nuclease, a gene or base editing protein, a prime editing protein, a Gene Writer protein (e.g., a modified or modularized non- long terminal repeat (LTR) retrotransposon), a retrotransposase, an RNA writer, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, a retrotransposon, a reverse transcriptase (e.g., M-MLV reverse transcriptase), a nickase or inactive nuclease (e.g., Cas9, nCas9, dCas9), a DNA recombinase, a CRISPR nuclease (e.g., Cas9, Cas12, Cas13, Cas3, CasMINI, Cas7-11, CasX), a DNA nickase, a Cas9 nickase (e.g., D10A or H840A), or any fusion or combination thereof. Other components include a guide RNA (gRNA), a single guide RNA (sgRNA), a prime editing guide RNA (pegRNA), a clustered regularly interspaced short palindromic repeat (CRISPR) RNA (crRNA), a trans-activating clustered regularly interspaced short palindromic repeat (CRISPR) RNA (tracrRNA), or a DNA molecule to be inserted or serve as a template for double-strand break (DSB) repair at a specific genomic locus. Genome-, gene-, and base- editing technology are reviewed in Anzalone et al., Nature Biotechnology 38:824-844, 2020, Sakuma, Gene and Genome Editing 3-4:100017, 2022, and Zhou et al., MedComm 3(3):e155, 2022, each of which is incorporated by reference for all that they teach about the components and uses of this technology to the extent that it does not conflict with this disclosure. [0041] “Conditioning agent,” as used herein, refers to a biological response modifier (BRM) that enhances the efficiency of engineering an immune cell, expands the number of immune cells available to be engineered or the number of engineered cells in a target tissue (for example, a tumor, fibrotic tissue, or tissue undergoing autoimmune attack), promotes activity of the engineered cell in a target tissue, or broadens the range of operative mechanisms contributing to a therapeutic immune reaction. A conditioning agent can be provided by delivering an encoding nucleic acid in a LNP or tLNP. Exemplary BRMs include cytokines, such as IL-7, IL-15, or IL-18. Conditioning agents can be provided as the agent itself or, when the conditioning agent is a peptide or protein, as a nucleic acid molecule encoding the conditioning agent. [0042] “Immune cell,” as used herein, can refer to any cell of the immune system. However, particular aspects can exclude polymorphonuclear leukocytes and/or B cells, or be limited to non-B lymphocytes such as T cell and/or NK cells, or to monocytes such as dendritic cells and/or macrophages in their various forms. [0043] As used herein, “lipid nanoparticle” (LNP) means a solid particle, as distinct from a liposome having an aqueous lumen. The core of an LNP, like the lumen of a liposome, is surrounded by a layer of lipid that can be, but is not necessarily, a continuous lipid monolayer, a bilayer, or multi-layer having three or more lipid layers. [0044] As used herein, a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide, peptide, carbohydrate, nucleic acid, or combination thereof that is capable of specifically binding to a target or multiple targets. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody, a Fab^, F(ab^)2, Fab, Fv, rIgG, scFv, hcAbs (heavy chain antibodies), a single domain antibody, VHH, VNAR, sdAbs, nanobody, receptor ectodomains or ligand-binding portions thereof, or ligands (e.g., cytokines, chemokines). A “Fab” (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond. In other embodiments, a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. A variety of assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, and Biacore® analysis. A binding moiety, such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains. [0045] As used herein, “antibody” refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen; so-called complementarity determining regions (CDRs). The term antibody can thus refer to intact or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (camels, alpacas, llamas, etc.) produce antibodies with both the canonical structure and antibodies comprising only heavy chains. The variable region of the camelid heavy chain only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody. Antigen binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH. Such elements can be combined to produce bi- and multi-specific reagents, such as T-cell engagers (for example, Bispecific T-Cell Engagers (BiTEs)) or other immune cell engagers. The term “monoclonal antibody” arose out of hybridoma technology but is now used to refer to any singular molecular species of antibody regardless of how it was originated or produced. Antibodies can be obtained through immunization, selection from a naïve or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that could be used as binding moieties are known in the art. An excellent source of information about antibodies for an International Non-proprietary Name (INN) has been proposed or recommended, including sequence information, is Wilkinson & Hale, 2022, MAbs 14(1):2123299, including its Supplementary Tables, which is incorporated by reference herein for all that it teaches about individual antibodies and the various antibody formats that can be constructed. U.S. Patent No.11,326,182 and especially its Table 9 entitled “Cancer, Inflammation and Immune System Antibodies,” is a source of sequence and other information for a wide range of antibodies including many that do not have an INN and is incorporated herein by reference for all that it teaches about individual antibodies. [0046] An antibody or other binding moiety (or a fusion protein thereof) “specifically binds” a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 105 M-1, while not significantly binding other components present in a test sample. Binding domains (or fusion proteins thereof) can be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Ka of at least 108 M-1, at least 109 M-1, at least 1010 M-1, at least 1011 M-1, at least 1012 M-1, or at least 1013 M-1, preferably at least 108 M-1 or at least 109 M-1. “Low affinity” binding domains refer to those binding domains with a Ka of up to 108 M-1, up to 107 M-1, up to 106 M-1, up to 105 M-1. Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5 M to 10-13 M). Affinities of binding domain polypeptides and fusion proteins according to this disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., 1949, Ann. N.Y. Acad. Sci.51:660; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent). [0047] As used herein, “payload” refers to a negatively charged agent that can interact with cationic lipids, such as the ionizable cationic lipids of this disclosure, to become encapsulated within lipid nanoparticles comprising the cationic lipid. The negatively charged agent can be a biologically active small organic molecule, or a macromolecule such as a nucleic acid molecule, a carbohydrate, a peptide, or a polypeptide. In some embodiments, a payload can be one or more nucleic acid molecules, RNA or DNA, including mRNA and guide RNA (gRNA) molecules. In certain embodiments, a payload comprises an mRNA that encodes a biologically active molecule, such as a chimeric antigen receptor, a T cell receptor, an immune cell engager, or the like. [0048] As used herein, “biologically active agent” refers to any substance that affects a metabolic or physiologic response in a living organism, cells, or cultured cells thereof, including genetically reprograming, permanently or transiently, such organism or cells. [0049] As used herein, “therapeutic agent” is a substance, or a component of a combination of substances, the biological activity of which can potentially cure, ameliorate, stabilize, prevent, or otherwise beneficially impact a disease, pathological condition, or other disorder. [0050] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, and the like). Nevertheless, such terms can also be used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g. CH3-CH2-), in certain circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term “alkylene.” (Similarly, in circumstances in which a divalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding divalent moiety, arylene.) All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for nitrogen, 2 for oxygen, and 2, 4, or 6 for sulfur, depending on the oxidation state of the sulfur atom). [0051] The term "alkyl" as employed herein refers to saturated straight and branched chain aliphatic groups having from 1 to 12 carbon atoms. As such, “alkyl” encompasses C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 groups. [0052] The term "alkenyl" as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms. As such, “alkenyl” encompasses C2, C3, C4, C5, C6, C7, C8, C9, C10, C11 and C12 groups. [0053] In some embodiments, the hydrocarbon chain is unsubstituted. In other embodiments, one or more hydrogens of the alkyl or alkenyl group can be substituted with the same or different substituents. [0054] Alkynoic refers to a carboxylic acid moiety comprising one or more carbon-carbon triple bonds. In some embodiments, hydrogens are unsubstituted. In other embodiments, one or more hydrogens of the alkynoic group can be substituted with the same or different substituents. [0055] Amide refers to a carboxylic acid derivative comprising a carbonyl group of a carboxylic acid bonded to an amine moiety. [0056] Ester refers to a carboxylic acid derivative comprising a carbonyl group bond to an alkyloxy group to form an ester bond -C(=O)-O-. [0057] Head group refers to the hydrophilic or polar portion of a lipid. [0058] Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group. Examples of sterols include, without limitation, cholesterol, ergosterol, ^-sitosterol, stigmasterol, stigmastanol, 20-hydroxycholesterol, 22-hydroxycholesterol, and the like. [0059] As standard, the bond represented as a solid wedge extends above the plane and the bond represented as a dashed wedge extends below the plane when depicting absolute stereochemistry, throughout. Ionizable Cationic Lipids [0060] In certain aspects, the ionizable cationic lipids of this disclosure have a structure of the formula M5: wherein: each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl, A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, A5 is absent, O, S, NH, or NCH3 if A4 is C=O, or A5 is C=O if A4 is not C=O, A6 is O, S, NH, NCH3 or (CH2)0-2, A7 is (CH2)0-6, wherein if A6 is O, S, NH, NCH3, A7 is (CH2)2-4, each W is independently CH or N; each R2 is independent O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when an R2 is O then its adjoining R3 is C=O, or when an R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of a W is O, that W is CH; and wherein when both R3 groups at a beta position of a W are C=O, that W is CH or N; wherein A6 and A7 are not both (CH2)0 unless A5 is C=O, or unless A4 is C=O and A5 is absent; wherein a) A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, or NCH3, A6 is (CH2)1-2, and A7 is (CH2)1-4, or b) A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, of O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4, or e) A1 is CH2, A3 is (CH2)1-5, X is CH, A4 is CH2, NH, NCH3 or O, A5 is C=O, A6 is Z N N (CH2)0-3CH3 (CH2)0, A7 is (CH2)0, and Y is , or f) A1 is (CH2)2, A3 is (CH2)1-5, X is CCH3, A4 is C=O, A5 is absent, A6 is (CH2)0, A7 is Z N N (CH2)0-3CH3 (CH2)0, and Y is ; wherein the number of contiguous atoms present in a span: the range from 7-17. [0061] The position of X can be referred to as the central branch point and the position of each W can be referred to as a distal branch point. Each of the disclosed ionizable cationic lipids has two tail groups extending from the central branch point and each tail group branches again at its distal branch point. [0062] As used herein, when a subscript has a value of “0”, the group is absent and the adjoining atoms are bounded to each other. For example, when A6 is (CH2)0, A6 is absent. [0063] In certain embodiments of formula M5 (including M5-1, M5-2, M5-3, and M5-4), A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, NCH3, A6 is (CH2)1-2, and A7 is (CH2)1-4. For example, in certain embodiments, A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, A6 is (CH2)1-2, and A7 is (CH2)1-4. [0064] In certain embodiments of formula M5, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, or O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NH, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M5, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NH, A5 is C=O, A6 is O, and A7 is (CH2)2-4. For example, in certain embodiments of formula M5, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NH, A5 is C=O, A6 is CH2, and A7 is (CH2)0-6. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, A5 is C=O, A6 is O, NH, NCH3, or CH2, or A7 is (CH2)0- 6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M5, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, A5 is C=O, A6 is O, and A7 is (CH2)2- 4. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M5, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is O, A5 is C=O, A6 is CH2, and A7 is (CH2)0-6. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NCH3, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M5, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NCH3, A5 is C=O, A6 is CH2, and A7 is (CH2)0-6. [0065] In certain embodiments of formula M5, A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, NH, NCH3, A6 is (CH2)1-2, or A7 is (CH2)1-4. For example, in certain embodiments, A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, A6 is (CH2)1-2, or A7 is (CH2)1-4. [0066] In certain embodiments of formula M5, the number of contiguous connective O A3 A4 A6 atoms present in a span: A1 A2 X A5 A7 is in the range from 7-17. For example, in certain embodiments, the number of contiguous connective atoms present in a span: O A3 A4 A6 A1 A2 X A5 A7 is in the range of 7-11 or 7-10. In certain embodiments, the O 3 4 6 number of contiguous connective atoms present in a span: A1 A A A2 X A5 A A7 is in the range of 10-17 (e.g., in the range of 10-16, or 10-14, or 10-12). For example, in certain embodiments, the number of contiguous connective atoms present in a span: O A3 4 6 A1 A2 A X A5 A A7 is 10. For example, in certain embodiments, the number of O A3 A4 A6 contiguous connective atoms present in a span: A1 A2 X A5 A7 is 7. The present inventors have found that changing the number of contiguous connective atoms present in each span can allow for tuning of the pKa of the cationic lipid. [0067] Different constituents for Y, A6, A7, and R1 allow for tuning of cLogD and c-pKa to achieve a target value of measured pKa within a LNP or a tLNP. For example, to make a CH3 (CH2)2 N lipid with a head group (comprising A6, A7, and Y) of CH3 less basic, the following head groups could be used instead: , [0068] Conversely, to make a lipid with a head group (comprising A6, A7, and Y) of CH3 (CH2)2 N CH3 more basic, the following head groups could be used instead: , [0069] The addition of CH2 groups in the head group (comprising A6, A7, and Y), will tend to increase basicity of the lipid which in turn will tend to increase measured pKa. The addition of CH2 groups in R1 will tend to increase the lipophilicity (cLogD) of the lipid which in turn will tend to decrease measured pKa of the LNP or tLNP. (CH2)0-3 CH 3 Z N [0070] In some embodiments of formula M5, Y is (CH2)0-3 CH 3 and Z is a bond. In (CH2)0-1 CH 3 Z N some embodiments of formula M5, Y is (CH2)0-1 CH 3 and Z is a bond. For example, in some embodiments of formula M5, Y is and Z is a bond. (CH2)2-4 OCH 3 Z N [0071] In some embodiments of formula M5, Y is (CH2)0-1 CH 3 and Z is a bond. For example, on some embodiments of formula bond. (CH2)2-4 OCH 3 Z N [0072] In some embodiments of formula M5, Y is (CH2)2-4 OCH 3 and Z is a bond. For example, in some embodiments of formula bond. [0073] In some embodiments of formula and Z is a bond. [0074] In some embodiments of formula M5, Y is and Z is a bond. [0075] some embodiments of formula M5, Y is and Z is a bond. [0076] some embodiments of formula M5, Y is and Z is a bond. Z N N (CH ) CH [0077] 2 0-3 3 In some embodiment of formula M5, Y is and Z is a bond. [0080] In some embodiments of formula M5, Y is and Z is a bond. [0081] In some embodiments of formula M5, Y is and Z is a bond. [0084] In some embodiments of formula M5, Y is and Z is a bond. [0087] In some embodiments of formula bond. [0092] As described above, in various embodiments of formula M5, each R1 is independently C7-C11 alkyl or C7-C11 alkenyl. In some embodiments of formula M5, each R1 is independently C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. In certain embodiments of formula M5, each R1 is independently a linear C7-C11 alkyl, e.g., a linear C7-C10 alkyl, or a linear C7-C9 alkyl. In some embodiments of formula M5 as described herein, each R1 is independently (CH2)6-8CH3. In some of these and other embodiments, R1 is (CH2)7CH3. In some embodiments of formula M5, each R1 is independently a linear C7-C11 alkenyl, e.g., a linear C7-C10 alkenyl, or a linear C7-C9 alkenyl. For example, in some embodiments of formula M5, each R1 is a linear C8 alkenyl. In certain other embodiments of formula M5, each R1 is independently a branched C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. For example, in some embodiments of formula M5, each R1 is a branched C8 alkyl. In certain embodiments of formula M5, each R1 is independently a branched C7-C11 alkenyl, e.g., C7- C10 alkenyl, or C7-C9 alkenyl. For example, in some embodiments of formula M5, each R1 is a branched C8 alkenyl. In some embodiments of formula M5, wherein R1 is a branched alkyl or alkenyl, the branch point is positioned so that ester carbonyls are not in an α position relative to the branch point, for example they are in a β position relative to the branch point. [0093] In certain embodiments of formula M5 as described herein, each R1 is the same. In certain embodiments of formula M5, each R1 nearest a common branch point is the same, but those nearest a first common branch point differ from those nearest a second common branch point. In certain embodiments of formula M5, each R1 nearest a common branch point is different but the pair of R1 groups nearest a first common branch point is the same the pair nearest a second common branch point. [0094] As described above, in various embodiments of formula M5 as described herein, each R2 is O or C=O, wherein at least one R2 is O and each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O. Accordingly, together, each R2 and R3 group form an ester group. In some embodiments of formula M5 as described herein, in each tail group one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O its adjoining R3 is O. In some embodiments of formula M5 as described herein, in one tail group, one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O its adjoining R3 is O, and in the other tail group, both R2 groups are O and both R3 groups are C=O. In some embodiments of formula M5 as described herein, in one tail group one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O its adjoining R3 is O, and in the other tail group both R2 groups are C=O and both R3 groups are O. In some embodiments of formula M5 as described herein, in one tail group, both R2 groups are O and both R3 groups are C=O, and in the other tail group both R2 groups are C=O and both R3 groups are O. In some embodiments of formula M5 as described herein, in each tail group both R2 groups are O and both R3 groups are C=O. [0095] As described above, each W is individually CH or N. For example, in some embodiments, each W is CH. In some embodiments, each W is N. In some other embodiments, one W is CH and the other W is N. In certain embodiments, when both R3 groups at a beta position of a W are C=O, that W can be CH or N. For example, in certain other embodiments, when both R3 groups at a beta position of a W are C=O, that W is N. In certain embodiments, when both R3 groups at a beta position of a W are C=O, that W is CH. [0096] When used in biological systems, it is advantageous to use ionizable cationic lipids that avoid formation of formaldehyde upon degradation. Accordingly, in certain embodiments as described herein, the combination of a R3 at a beta position of W being O and W being N is avoided. For example, in other certain embodiments, when at least one R3 at a beta position of a W is O, that W is CH. In other certain embodiments, when both R3 groups at a beta position of a W are O, that W is CH. [0097] As described above, in some embodiments of formula M5 as described herein, in each tail group one R2 is O and the other R2 is C=O, and one R3 is C=O and the other R3 is O, respectively. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-1: wherein A1, A2, A3, X, A4, A5, A6, A7, Y, and R1 are as otherwise described herein. [0098] As described above, in some embodiments of formula M5 as described herein, in one tail group one R2 is O and the other R2 is C=O, and one R3 is C=O and the other R3 is O, respectively, and in the other tail group both R2 groups are C=O and both R3 groups are O. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-2: wherein A1, A2, A3, X, A4, A5, A6, A7, Y, and R1 are as otherwise described herein. [0099] As described above, in some embodiments of formula M5 as described herein, in one tail group, one R2 is O and the other R2 is C=O, and one R3 is C=O and the other R3 is O, respectively, and in the other tail group, both R2 groups are O and both R3 groups are C=O. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-3: wherein A1, A2, A3, X, A4, A5, A6, A7, Y, W, and R1 are as otherwise described herein. [00100] As described above, in some embodiments of formula M5 as described herein, in one tail group, both R2 groups are O and both R3 groups are C=O, and in the other tail group, both R2 groups are C=O and both R3 groups are O. Accordingly, in certain aspects, the constrained ionizable cationic lipids of this disclosure have a structure of formula M5-4: wherein A1, A2, A3, X, A4, A5, A6, A7, Y, W, and R1 are as otherwise described herein. [00101] In certain aspects of the ionizable cationic lipids of formula M5 as described herein, in both tail groups, both R2 groups are O and both R3 groups are C=O. For example, in certain aspects, the ionizable cationic lipids of this disclosure have a structure of the formula M4: wherein: each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl, A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 is X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, A5 is absent, O, S, NH, NCH3, or C=O, A6 is O, S, NH, NCH3 or (CH2)0-2, A7 is (CH2)0-6, wherein if A6 is O, S, NH, NCH3, A7 is (CH2)2-4, and W is CH or N; wherein A6 and A7 are not both (CH2)0 unless A5 is C=O, or unless A4 is C=O and A5 is absent; wherein a) A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, or NCH3, A6 is (CH2)1-2, and A7 is (CH2)1-4, or b) A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, or O, A5 is C=O, A6 is O, NH, NCH3, or CH2, A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, and A7 is (CH2)2-4 or e) A1 is CH2, A3 is (CH2)1-5, X is CH, A4 is CH2, NH, NCH3 or O, A5 is C=O, A6 is 2 0 7 Z N N (CH2)0-3CH3 (CH ) , A is (CH2)0, and Y is , or f) A1 is (CH2)2, A3 is (CH2)1-5, X is CCH3, A4 is C=O, A5 is absent, A6 is (CH2)0, A7 is Z N N (CH2)0-3CH3 (CH2)0, and Y is ; wherein the number of contiguous atoms present in a span: the range from 7-17. [00102] As used herein, when a subscript has a value of “0”, the group is absent. For example, when A6 is (CH2)0, A6 is absent. [00103] In certain embodiments of formula M4, A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, NCH3, A6 is (CH2)1-2, and A7 is (CH2)1-4. For example, in certain embodiments A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, A6 is (CH2)1-2, and A7 is (CH2)1-4. [00104] In certain embodiments of formula M4, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, or O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NH, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M4, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NH, A5 is C=O, A6 is O, and A7 is (CH2)2-4. For example, in certain embodiments of formula M4, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NH, A5 is C=O, A6 is CH2, and A7 is (CH2)0-6. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, A5 is C=O, A6 is O, NH, NCH3, or CH2, or A7 is (CH2)0- 6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M4, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, A5 is C=O, A6 is O, A7 is (CH2)2-4. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M4, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is O, A5 is C=O, A6 is CH2, and A7 is (CH2)0-6. In certain embodiments as described herein, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NCH3, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. For example, in certain embodiments of formula M4, A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is NCH3, A5 is C=O, A6 is CH2, and A7 is (CH2)0-6. [00105] In certain embodiments of formula M4, A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, NH, NCH3, A6 is (CH2)1-2, or A7 is (CH2)1-4. For example, in certain embodiments, A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, A6 is (CH2)1-2, or A7 is (CH2)1-4. [00106] In certain embodiments of formula M4, the number of contiguous connective O A3 A4 A6 atoms present in a span: A1 A2 X A5 A7 is in the range from 7-17. For example, in certain embodiments, the number of contiguous connective atoms present in a span: O is in the range of 7-11 or 7-10. In certain embodiments, the number O of contiguous connective atoms present in a span: is in the range of 10-17 (e.g., in the range of 10-16, or 10-14, or 10-12). For example, in certain embodiments, the number of contiguous connective atoms present in a span: O A3 A4 A6 A1 A2 X A5 A7 is 10. For example, in certain embodiments, the number of contiguous connective atoms present in a span: is 7. As set forth herein, changing the number of contiguous connective atoms present in each span can allow for tuning of the pKa of the cationic lipid. [00107] Different constituents for Y, A6, A7, and R1 allow for tuning of cLogD and c-pKa to achieve a target value of measured pKa within a LNP or a tLNP. For example, to make a CH3 (CH2)2 N lipid with a head group (comprising A6, A7, and Y) of CH3 less basic, the following N N (CH2)0-3CH3 (CH2)2 N OH head groups could be used instead: , , [00108] Conversely, to make a lipid with a head group (comprising A6, A7, and Y) of CH3 (CH2)2 N CH3 more basic, the following head groups could be used instead: , [00109] The addition of CH2 groups in the head group (comprising A6, A7, and Y), will tend to increase basicity of the lipid which in turn will tend to increase measured pKa. The addition of CH2 groups in R1 will tend to increase the lipophilicity (cLogD) of the lipid which in turn will tend to decrease measured pKa of the LNP or tLNP. (CH2)0-3 CH 3 Z N [00110] In some embodiments of formula M4, Y is (CH2)0-3 CH 3 and Z is a bond. In (CH2)0-1 CH 3 Z N some embodiments of formula M4, Y is (CH2)0-1 CH 3 and Z is a bond. For example, in Z N some embodiments of formula M4, Y is and Z is a bond. (CH2)2-4 OCH 3 Z N [00111] In some embodiments of formula M4, Y is (CH2)0-1 CH 3 and Z is a bond. For example, on some embodiments of formula bond. (CH2)2-4 OCH 3 Z N [00112] In some embodiments of formula M4, Y is (CH2)2-4 OCH 3 and Z is a bond. For example, in some embodiments of formula bond. [00113] In some embodiments of formula and Z is a bond. [00114] In some embodiments of formula and Z is a bond. Z N O [00115] In some embodiments of formula M4, Y is and Z is a bond. [00116] In some embodiments of formula and Z is a bond. Z N N (CH2)0-3CH3 [00117] In some embodiment of formula M4, Y is and Z is a bond. [00120] In some embodiments of formula M4, Y is and Z is a bond. [00121] In some embodiments of formula M4, Y is and Z is a bond. [00124] In some embodiments of formula M4, Y is and Z is a bond. [00127] In some embodiments of formula bond. [00128] In some embodiments of formula bond. [00129] In some embodiments of formula [00132] As described above, in some embodiments of formula M4, it can be desirable to decrease the basicity of the ionizable cationic lipid by selecting an appropriate Y group as Z N N (CH2)0-3CH3 described herein. One such Y group is . Accordingly, in some embodiments of formula (CH2)0, A7 is (CH2)0, and [00133] In some embodiments of formula M4, A1 is CH2, A3 is (CH2)1-5, X is CH, A4 is CH2, Z N N (CH2)0-3CH3 NH, NCH3 or O, A5 is C=O, A6 is (CH2)0, A7 is (CH2)0, and Y is . For example, in some embodiments, A1 is CH2, A3 is (CH2)1-5, X is CH, A4 is NH, or O, A5 is C=O, 6 2 0 7 Z N N (CH2)0-3CH3 A is (CH ) , A is (CH2)0, and Y is . For example, in some embodiments, A1 is CH2, A3 is (CH2)1-5, X is CH, A4 is CH2, A5 is C=O, A6 is (CH2)0, A7 is Z N N (CH2)0-3CH3 (CH2)0, and Y is . For example, in some embodiments, A1 is CH2, A3 is (CH2)1-5, X is CH, A4 is O, A5 is C=O, A6 is (CH2)0, A7 is (CH2)0, and Y is Z N N (CH2)0-3CH3 . For example, in some embodiments, A1 is CH2, A3 is (CH2)1-5, X is [00134] In some embodiments of formula M4, A1 is (CH2)2, A3 is (CH2)1-5, X is CCH3, A4 is Z N A5 is absent, A6 N (CH2)0-3CH3 C=O, is (CH2)0, A7 is (CH2)0, and Y is . [00135] As described above, in various embodiments of formula M4, each R1 is independently C7-C11 alkyl or C7-C11 alkenyl. In some embodiments of formula M4, each R1 is independently C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. In certain embodiments of formula M4, each R1 is independently a linear C7-C11 alkyl, e.g., a linear C7-C10 alkyl, or a linear C7-C9 alkyl. In some embodiments of formula M4 as described herein, each R1 is independently (CH2)6-8CH3. In some of these and other embodiments of formula M4, R1 is (CH2)7CH3. In some embodiments of formula M4, each R1 is independently a linear C7-C11 alkenyl, e.g., a linear C7-C10 alkenyl, or a linear C7-C9 alkenyl. For example, in some embodiments of formula M4, each R1 is a linear C8 alkenyl. In certain other embodiments of formula M4, each R1 is independently a branched C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. For example, in some embodiments of formula M4, each R1 is a branched C8 alkyl. In certain embodiments of formula M4, each R1 is independently a branched C7-C11 alkenyl, e.g., C7-C10 alkenyl, or C7-C9 alkenyl. For example, in some embodiments of formula M4, each R1 is a branched C8 alkenyl. In some embodiments of formula M4, wherein R1 is a branched alkyl or alkenyl, the branch point is positioned so that ester carbonyls are not in an α position relative to the branch point, for example they are in a β position relative to the branch point. [00136] In certain embodiments of formula M4 as described herein, each R1 is the same. In certain embodiments of formula M4, each R1 nearest a common branch point is the same, but those nearest a first common branch point differ from those nearest a second common branch point. In certain embodiments of formula M4, each R1 nearest a common branch point is different but the pair of R1 groups nearest a first common branch point is the same the pair nearest a second common branch point. [00137] As described above, W is CH or N. In certain embodiments of formula M4, Z is CH. In certain other embodiments of formula M4, Z is N. [00138] Ionizable cationic lipids as described herein, can be useful as a component of lipid nanoparticles for delivering nucleic acids, including DNA, mRNA, or siRNA into cells. The ionizable cationic lipids can have a c-pKa (calculated pKa) in the range of from about 6, 7, or 8 to about 9, 10, or 11. For example, in various embodiments as described herein, the ionizable cationic lipids have a c-pKa ranging from about 6 to about 10, about 7 to about 10, about 8 to about 10, about 8 to about 9, 6 to 10, 7 to 10, 8 to 10, or 8 to 9. In certain embodiments, the ionizable cationic lipids have a c-pKa ranging from about 8.4 to about 8.7 or 8.4 to 8.7. The ionizable cationic lipids as described herein can have cLogD ranging from about 9 to about 18, for example, ranging from about 10 to about 18, or about 10 to about 16, to about 10 to about 14, or about 11 to about 18, or about 11 to about 15, or about 11 to about 14, or about 12 to about 14. The ionizable cationic lipids as described herein can have cLogD ranging from 9 to 18, for example, ranging from 10 to 18, or 10 to 16, to 10 to 14, or 11 to 18, or 11 to 15, or 11 to 14, or 12 to 14. In certain embodiments, the ionizable cationic lipids have a cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4. In certain embodiments, the ionizable cationic lipids as described herein can have a c-pKa ranging from about 8 to about 11 or from 8 to 11 and a cLogD ranging from about 9 to about 18 or from 9 to 18. For example, in certain embodiments, the ionizable cationic lipids have a c- pKa ranging from about 8.4 to about 8.7 or from 8.4 to 8.7 and cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4. These ranges can lead to a measured pKa in the LNP ranging from about 6 to about 7 or from 6 to 7, which facilitates ionization in an endosome after delivery into a cell. [00139] Ionizable cationic lipids of this disclosure have a branched structure to give the lipid a conical rather than cylindrical shape and such structure helps promote endosomolytic activity when incorporated into an LNP. The greater the endosomolytic activity, the more efficient is release of the biologically active payload (e.g., one or more species of nucleic acid molecules). [00140] Another consideration in designing or selecting a particular ionizable cationic lipid for use in an LNP for delivering nucleic acids (or other negatively charged payloads) into cells is the type of cell and, for in vivo delivery, locus within the body targeted for delivery. This is also influenced by pKa. Zhang et al. (iScience 27:109804, 2024) relates that measured pKa >9 can favor accumulation in the lungs, measured pKa of 2-6 can favor accumulation in spleen, and measured pKa of 6-7 can favor accumulation in liver. Thus, ionizable lipids with pKa across a wide range can be useful for delivery to various tissues. Of course, other factors can also impact what tissue or cells an LNP will deliver its payload to, including the use of a targeting moiety attached to the surface of the LNP. [00141] In some embodiments, somewhat greater basicity can be desirable and can be obtained from ionizable cationic lipids with c-pKa and cLogD in the ranges disclosed herein. In some embodiments, cLogD of ionizable cationic lipids of this disclosure is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or in a range bound by any pair of these values. Lipid design also accounts for potential biodegradability pathways of target lipids, such as by way of esterases in plasma, liver and other tissues. Another consideration in lipid design is the fate of fragments of ionizable lipids resulting from degradation, such as after esterase cleavage(s). Preferably, the resulting fragments are rapidly cleared from the body without the need for hepatic oxidative metabolism. [00142] As used herein, cLogD is a calculated measure of lipophilicity that accounts for the state of ionization of the lipid molecule at a particular pH, which is a predictor of partitioning of the lipid between water and octanol as a function of pH. More specifically, cLogD is calculated at a specified pH based on cLogP and c-pKa. (LogP is the partition coefficient of a lipid molecule between aqueous (e.g., water) and lipophilic (e.g., octanol) phases). Numerous software packages are available to calculate cLogD values. When higher basicity of an ionizable lipid is desired, it should be balanced by greater lipophilicity as represented by a higher cLogD value. Balance of basicity and lipophilicity is needed for optimal functioning of the LNP for both stability of the particle and release of the biologically active payload (e.g., one or more species of nucleic acid molecules that can encode a therapeutic agent) upon uptake by a cell. Accordingly, for ionizable cationic lipids of this disclosure having a structure of formula M4, as R1 increases from C6-C10, the increase overall lipophilicity of the ionizable cationic lipid will increase, as represented by cLogD. This can be balanced by alterations in the head group (comprising A6, A7, and Y) as discussed above which result in higher c-pKa based on the basicity of the head group. Each of the ionizable cationic lipid species described herein have a cLogD and c-pKa values within the desired range(s), as described herein. Specific cLogD and c-pKa values have been calculated using ACD Labs Structure Designer v 12.0 for ionic cationic lipids of the disclosure. cLogP was calculated using ACD Labs Version B; cLogD was calculated at pH 7.4. Table 1 shows cLogD and c-pKa for CICL-250 and CICL-291– CICL-303. Table 1. cLogD and c-pKa of Exemplary CICL [00143] In some embodiments, the ionizable cationic lipid has the structure CICL-250: CICL-250 [00144] In some embodiments, the ionizable cationic lipid has the structure CICL-291: [00145] In some embodiments, the ionizable cationic lipid has the structure CICL-292: [00146] In some embodiments, the ionizable cationic lipid has the structure CICL-293: [00147] In some embodiments, the ionizable cationic lipid has the structure CICL-294: [00148] In some embodiments, the ionizable cationic lipid has the structure CICL-295: [00149] In some embodiments, the ionizable cationic lipid has the structure CICL-296: [00150] In some embodiments, the ionizable cationic lipid has the structure CICL-297: [00151] In some embodiments, the ionizable cationic lipid has the structure CICL-298: [00152] In some embodiments, the ionizable cationic lipid has the structure CICL-299: [00153] In some embodiments, the ionizable cationic lipid has the structure CICL-300: [00154] In some embodiments, the ionizable cationic lipid has the structure CICL-301: [00155] In some embodiments, the ionizable cationic lipid has the structure CICL-302: [00156] In some embodiments, the ionizable cationic lipid has the structure CICL-303: [00157] In some embodiments, the ionizable cationic lipid has the structure CICL-250-61: [00158] In some embodiments, the ionizable cationic lipid has the structure CICL-250-62: [00159] In some embodiments, the ionizable cationic lipid has the structure CICL-250-63: [00160] In some embodiments, the ionizable cationic lipid has the structure CICL-250-64: [00161] In some embodiments, the ionizable cationic lipid has the structure CICL-250-65: [00162] In some embodiments, the ionizable cationic lipid has the structure CICL-250-66: [00163] In some embodiments, the ionizable cationic lipid has the structure CICL-250-67: [00164] In some embodiments, the ionizable cationic lipid has the structure CICL-250-68: [00165] In some embodiments, the ionizable cationic lipid has the structure CICL-250-69: [00166] In some embodiments, the ionizable cationic lipid has the structure CICL-250-70: [00167] In some embodiments, the ionizable cationic lipid has the structure CICL-250-71: [00168] In some embodiments, the ionizable cationic lipid has the structure CICL-250-72: [00169] In some embodiments, the ionizable cationic lipid has the structure CICL-250-73: [00170] In some embodiments, the ionizable cationic lipid has the structure CICL-250-74: [00171] In some embodiments, the ionizable cationic lipid has the structure CICL-250-75: [00172] In some embodiments, the ionizable cationic lipid has the structure CICL-250-76: [00173] In some embodiments, the ionizable cationic lipid has the structure CICL-250-77: [00174] In some embodiments, the ionizable cationic lipid has the structure CICL-309: [00175] In some embodiments, the ionizable cationic lipid has the structure CICL-310: [00176] In some embodiments, the ionizable cationic lipid has the structure CICL-311: CICL-311 [00177] In some embodiments, the ionizable cationic lipid has the structure of the following: [00178] In other aspects of this disclosure are provided intermediate lipids of the ionizable cationic lipids disclosed herein. In some embodiments, the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M5 has the structure of formula I5-1: wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; each R2 is independent O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=O, W is CH or N; A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, and R4 is H or a protecting group. [00179] In various embodiments as described herein, R4 is H. [00180] In various embodiments as described herein, R4 is a protecting group (i.e., PG1). PG1 can be selected from base labile or acid labile protecting groups as known in the art. For example, in some embodiments, R4 is an acid labile protecting group such as t-butoxycarbonyl (BOC) or benzyloxycarbonyl (Cbz). In some other embodiments, R4 is a base labile protecting group such as a trimethylsilylethoxycarbonyl moiety. [00181] In certain embodiments of formula I5-1, when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. In certain embodiments of I5-1, when A1 and A3 are CH2, then X is CH, and A4 is NH2. [00182] In certain embodiments of formula I5-1, when A1 is CH2 and A3 is CH2 or CH2CH2, and X is CH, then A4 is NH, NCH3, or O. In certain embodiments of formula I5-1, when A1 is CH2, A3 is CH2CH2, X is CH, and A4 is O. In certain embodiments of formula I5-1, when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NH. In certain embodiments of formula I5-1, when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NCH3. [00183] In some embodiments, the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M5 has the structure of formula I5-2: wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; each R2 is independent O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=O, W is CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, and A4 is CH2, NH, NCH3, or O, and [00184] In various embodiments of formula I5-2, R5 is OH. [00185] In various embodiments of formula I5-2, R5 is . [00186] In certain embodiments of formula I5-2, when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. [00187] In certain embodiments of formula I5-2, A1 and A3 are CH2, X is CH, A4 is NH2, and [00188] In certain embodiments of formula I5-2, A1 and A3 are CH2, X is CH, A4 is CH2, and R5 is OH. [00189] In some embodiments, the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M5 has the structure of formula I5-3:
wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; each R2 is independent O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=O, W is CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, and X is N, CH, or C-CH3; and [00190] In various embodiments of formula I5-3, R6 is OH. [00191] In various embodiments of formula I5-3, R6 is . [00192] In certain embodiments of formula I5-3, A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is C-CH3. [00193] In certain embodiments of formula I5-3, A1 is CH2, A3 is CH2CH2, X is C-CH3, and R6 is OH. [00194] In certain embodiments of formula I5-3, A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is N. [00195] In certain embodiments of formula I5-3, A1 is CH2, A2 is O, A3 is CH2CH2, X is N, [00196] In other aspects of this disclosure are provided intermediate lipids of the ionizable cationic lipids disclosed herein. In some embodiments, the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M4 has the structure of formula I4-1: wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, and R4 is H or a protecting group. [00197] In various embodiments as described herein, R4 is H. [00198] In various embodiments as described herein, R4 is a protecting group (i.e., PG1). PG1 can be selected from base labile or acid labile protecting groups as known in the art. For example, in some embodiments, R4 is an acid labile protecting group such as t-butoxycarbonyl (BOC) or benzyloxycarbonyl (Cbz). In some other embodiments, R4 is a base labile protecting group such as a trimethylsilylethoxycarbonyl moiety. [00199] In certain embodiments of formula I4-1, when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. In certain embodiments of I4-1, when A1 and A3 are CH2, then X is CH, and A4 is NH2. [00200] In certain embodiments of formula I4-1, when A1 is CH2 and A3 is CH2 or CH2CH2, and X is CH, then A4 is NH, NCH3, or O. In certain embodiments of formula I4-1, when A1 is CH2, A3 is CH2CH2, X is CH, and A4 is O. In certain embodiments of formula I4-1, when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NH. In certain embodiments of formula I4-1, when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NCH3. [00201] In some embodiments, the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M4 has the structure of formula I4-2: wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, and A4 is CH2, NH, NCH3, or O, and [00202] In various embodiments of formula I4-2, R5 is OH. [00203] In various embodiments of formula I4-2, R5 is . [00204] In certain embodiments of formula I4-2, when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. [00205] In certain embodiments of formula I4-2, A1 and A3 are CH2, X is CH, A4 is NH2, and [00206] In certain embodiments of formula I4-2, A1 and A3 are CH2, X is CH, A4 is CH2, and R5 is OH. [00207] In some embodiments, the lipid (e.g., intermediate lipid) of the ionizable cationic lipid of formula M4 has the structure of formula I4-3: wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, and X is N, CH, or C-CH3; and [00208] In various embodiments of formula I4-3, R6 is OH. [00209] In various embodiments of formula I4-3, R6 is . [00210] In certain embodiments of formula I4-3, A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is C-CH3. [00211] In certain embodiments of formula I4-3, A1 is CH2, A3 is CH2CH2, X is C-CH3, and R6 is OH. [00212] In certain embodiments of formula I4-3, A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is N. [00213] In certain embodiments of formula I4-3, A1 is CH2, A2 is O, A3 is CH2CH2, X is N, [00214] To promote biodegradability and minimize accumulation of ionizable cationic lipids of this disclosure, the fatty acid tails are designed to comprise esters in a position that minimizes steric hindrance of ester cleavage. For example, while a single fatty acid tail will tend to extend away from the ester carbonyl to provide the most energetically favorable position, the presence of two tails leads to the tails extending in opposite directions to provide the most energetically favorable conformation. Conformation is dynamic and the fatty acid tails can also move through less energetically favorable positions. For example, in certain embodiments, one of the tails can extend toward the carbonyl and sterically hinders cleavage of the ester. Accordingly, large branches immediately adjacent to the ester carbonyl were avoided in the design of the ionizable cationic lipids disclosed herein. For example, in some embodiments, there is at least one atom between the ester carbonyl of a tail group and the branch point (i.e., W, as described herein). In certain embodiments, there are at least two atoms between the ester carbonyl of the tail group and the branch point (i.e., W, as described herein). [00215] In positioning the ester(s) within the lipid, consideration was also given to potential degradation products to avoid generation of toxic compounds, such as formaldehyde, when W is N. Additionally, to promote biodegradability, in formulas M5 and M4, A1 is (CH2)1-2 and A2 is oxygen and formulas M4 and M5 do not encompass lipids where A1 is oxygen and A2 is (CH2)1-2. (That is, the ester including the carbonyl between A1 and A2 is oriented so that its carbonyl is closer to the nearest W than is its O.) Without being bound by, theory, it will be recognized by the skilled artisan that having a CH2 or a (CH2)2 at the A1 position advantageously permits for any intramolecular transesterification to lead to further degradation of the ionizable cationic lipid, whereas if A1 were oxygen such a transesterification would be a simple ester exchange and not release any part of the molecule. [00216] The configuration of the esters around W also can affect the rate and pathway of biodegradation of the lipid. When an R3 is O, esterase-mediated cleavage of the ester releases a carboxylic acid and a hydroxyl remains which can cyclize in an intramolecular transesterification forming a butyrolactone and releasing the core structure, a reaction known to proceed rapidly. For this mechanism to proceed it is important that the anteriorly located esters (i.e., the esters nearest to the central branchpoint nitrogen atom) not be oriented with its O closer to W than its C=O, as in such a structure the transesterification would be a simple intramolecular ester exchange and no piece of the molecule would be released. Accordingly, to promote biodegradation, it is advantageous for a methylene to be at an alpha position of W. [00217] An advantage of ionizable cationic lipids of this disclosure is that, at least in part, the toxicity associated with quaternary ammonium cationic lipids can be avoided. Some LNPs containing quaternary ammonium lipids, which are effectively permanently cationic, have displayed a fatal hyperacute toxicity in laboratory animals. In contrast, the use of ionizable cationic lipids of this disclosure in an LNP obviates the need for quaternary ammonium cationic lipids and, thereby, can mitigate or avoid potential LNP toxicity. In certain embodiments, use of an LNP or tLNP of this disclosure causes no detectable toxicity to cells or in a subject. In certain embodiments, use of an LNP or tLNP of this disclosure causes no more than mild toxicity to cells or in a subject that is asymptomatic or induces only mild symptoms that do not require intervention. In certain embodiments, use of an LNP or tLNP of this disclosure causes no more than moderate toxicity to cells or in a subject which may impair activities of daily living that requires only minimal, local, or non-invasive interventions. [00218] The relationship between the efficacy and toxicity of a drug is generally expressed in terms of therapeutic window and therapeutic index. Therapeutic window is the dose range from the lowest dose that exhibits a detectable therapeutic effect up to the maximum tolerated dose (MTD); the highest dose that will the desired therapeutic effect without producing unacceptable toxicity. Most typically, therapeutic index is calculated as the ratio of LD50:ED50 when based on animal studies and TD50:ED50 when based on studies in humans (though this calculation could also be derived from animal studies and is sometimes called the protective index), where LD50, TD50, and ED50 are the doses that are lethal, toxic, and effective in 50% of the tested population, respectively. These concepts are applicable whether the toxicity is based on the active agent itself or some other component of the drug product, such as, for example, the LNP or its components. For any inherent level of toxicity of the disclosed lipids or LNPs themselves, an increase in the efficiency of delivering the nucleic acid into the cytoplasm will improve the therapeutic window or index, as an effective amount of a biologically active payload (e.g., one or more species of nucleic acid molecule) would be deliverable with a smaller dosage of LNP (and its component lipids). [00219] Toxicities and adverse events can be graded according to a 5-point scale. A grade 1 or mild toxicity is asymptomatic or induces only mild symptoms; may be characterized by clinical or diagnostic observations only; and intervention is not indicated. A grade 2 or moderate toxicity may impair activities of daily living (such as preparing meals, shopping, managing money, using the telephone, etc.) but only minimal, local, or non- invasive interventions are indicated. Grade 3 toxicities are medically significant but not immediately life-threatening; hospitalization or prolongation of hospitalization is indicated; activities of daily living related to self-care (such as bathing, dressing and undressing, feeding oneself, using the toilet, taking medications, and not being bedridden) may be impaired. Grade 4 toxicities are life-threatening and urgent intervention is indicated. Grade 5 toxicity produces an adverse event-related death. Thus, in various embodiments, by use of the disclosed LNP and tLNP a toxicity is confined to grade 2 or less, grade 1 or less, or produces no observed toxicity. Tolerability [00220] Conventional LNPs deliver primarily to the liver. Liver toxicity has been the major dose- limiting parameter observed with LNP-containing pharmaceuticals. For example, ONPATTRO®, comprising the ionizable lipid MC3, has a NOAEL (no observed adverse effect level) of only 0.3 mg/kg for multiple dosing in rats. A benchmark LNP comprising the ionizable cationic lipid ALC-0315, used in the SARS-CoV-2 vaccine COMIRNATY®, caused elevated levels of liver enzymes and acute phase proteins at single doses of ≥1 mg/kg in the rat. Merely attaching an antibody to the benchmark LNP partially reverses that elevation and the reversal is greater if the antibody directs the LNP to some other tissue (that is, a tLNP). However, use of a highly biodegradable ionizable cationic lipid, CICL-1, similar to those disclosed herein, reduced delivery to the liver and associated liver enzyme and acute phase protein levels to a greater extent for LNP, antibody-conjugated LNP, and tLNP. Methods of Making Ionizable Cationic Lipids [00221] Structural symmetries and convergent rather than linear synthesis pathways can be used to simplify the synthesis of the ionizable lipids of this disclosure. [00222] In certain aspects, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M5 (e.g., CICL-309, CICL-310, and CICL-311). [00223] In certain aspects, this disclosure provides methods for synthesizing an ionizable cationic lipid of formula M4 (e.g., CICL-250, CICL-291, CICL-292, CICL-293, CICL-294, CICL-295, CICL-296, CICL-297, CICL-298, CICL-299, CILC-300, CICL-301, CICL-302, and CICL-303). [00224] Table 2 provides a summary of substituents in formula M4 of CICL-250 and CICL-291 – CICL-303.
Table 2. Exemplary CICL Structures [00225] Synthesis of an ionizable cationic lipid of formula CICL-250, CICL-297, CICL-291, and CICL-298 [00226] In certain embodiments, this disclosure provides a method of synthesizing an ionizable cationic lipid of formula CICL-250, CICL-297, CICL-291 and CICL-298 comprising the synthesis step as shown in Scheme 1, wherein the methylated acylimidazolide is reacted with HB in the presence of a mild base such as Et3N or Me3N and HB is an alcohol, amine, or thiol,, and the rest of the substituents are defined the same as in formulas CICL- 250, CICL-297, CILC-291, and CICL-298. when X = CH and A4 = NH Scheme 1 [00227] The synthesis step shown in Scheme 1 comprises reacting a diester-X-amine (1- A or 1B) with 1,1'-carbonyldiimidazole (CDI) to provide an imidazolecarboxyamide (1-C or 1- D); and after activation of the imidazolecarboxyamide (1-C or 1-D) it is coupled with a desired B (i.e. HB) to provide 1-E, wherein B is A5-A6-A7-Y, or 1-F, wherein B is A6-A7-Y. For 1-E, all substituents are defined the same as those in formula CICL-250 or CICL-297. For 1-F, all substituents are defined the same as those in formula CICL-291 or CICL-298. In certain embodiments, the imidazolecarboxyamide 1-C or 1-D synthesis is carried out in an organic solvent (e.g., without limitation, CH2Cl2) in the presence of a basic catalyst (e.g., without limitation, Et3N). In certain embodiments, the synthesis can comprise first reacting the imidazolecarboxyamide 1-C or 1-D with MeOTf, then reacting with the desired B (i.e. HB) in the presence of a base (e.g., trimethylamine). The reaction can be carried out in an organic solvent (e.g., acetonitrile). [00228] Synthesis of an ionizable cationic lipid of formula CICL-292 and CICL-299 [00229] In certain embodiments, this disclosure provides a method of synthesizing an ionizable cationic lipid of formula CICL-292 and CICL-299 comprising the synthesis step shown in Scheme 2, all substituents are defined the same as in formula CICL-292 or CICL 299. Scheme 2 [00230] The synthesis step shown in Scheme 2 comprises coupling a diester-X-A5-acid 2- A with an amine/alcohol (i.e. HB) to provide an amide/ester having the structure of formula 2B, wherein B is A6-A7-Y. In certain embodiments, the coupling reaction is carried out in an organic solvent (e.g., THF) in the presence of basic catalysts (e.g., EDC-HCl and DMAP, or HATU and i-Pr2NEt). [00231] Synthesis of an ionizable cationic lipid of formula CICL-293, CICL-294, CICL- 295, CILC-300, CICL-301, CICL-302, CICL-296, and CICL-303 [00232] In some embodiments, the ionizable cationic lipid of formula M4 synthesized has a structure of formula CICL-293, CICL-294, CICL-295, CILC-300, CICL-301, CICL-302, CICL- 296, and CICL-303. The method comprises the synthesis steps shown in Scheme 3.
Scheme 3 [00233] The synthesis step shown in Scheme 3 comprises coupling the diester-X- amine/alcohol (3A or 3B) with a desired base, HOOC-B or HO-B, to provide an amide/ester having the structure of formula 3-C, wherein B is A6-A7-Y, or 3-D, wherein B is A6-A7-Y. In certain embodiments, the coupling reaction is carried out in an organic solvent (e.g., acetonitrile, THF, or CH2Cl2) in the presence of a nucleophilic catalyst (e.g., DMAP), an acidic catalyst (e.g., EDC-HCl) and a basic catalyst (e.g., Et3N). [00234] Synthesis of an ionizable cationic lipid of formula CICL-309, CICL-310, and CICL-311 [00235] In certain embodiments, this disclosure provides a method of synthesizing an ionizable cationic lipid of formula CICL-309, CICL-310, and CICL-311 comprising the synthesis steps as shown in Scheme 4 wherein the methylated acylimidazolide is reacted with HB in the presence of a mild base such as Et3N or Me3N and HB is an alcohol, amine, or thiol, and the rest of the substituents are defined the same as in formulas CICL-309, CICL-310, and CICL-311. Scheme 4 [00236] The synthesis step shown in Scheme 4 comprises reacting a diester-X-amine (4- A) with 1,1'-carbonyldiimidazole (CDI) to provide an imidazolecarboxyamide (4-C); and after activation of the imidazolecarboxyamide (4-C) it is coupled with a desired B (i.e. HB) to provide 4-E, wherein B is A5-A6-A7-Y. For 1-E, all substituents are defined the same as those in formula CICL-309, CICL-310, or CICL-311. In certain embodiments, the imidazolecarboxyamide 4-C synthesis is carried out in an organic solvent (e.g., without limitation, CH2Cl2) in the presence of a basic catalyst (e.g., without limitation, Et3N). In certain embodiments, the synthesis can comprise first reacting the imidazolecarboxyamide 4-C with MeOTf, then reacting with the desired B (i.e. HB) in the presence of a base (e.g., trimethylamine). The reaction can be carried out in an organic solvent (e.g., acetonitrile). [00237] As described above, in various embodiments as described herein HB is selected from H-A6-A7-Y, H-A5-A6-A7-Y, and HOOC-A6-A7-Y are used under coupling conditions to provide the ionizable cationic lipids of formulas M5 and M4 as described herein. In various embodiments as described herein, H-A6-A7-Y is selected from the group consisting of A6 and A7 are as otherwise described herein. The various H-A6-A7-Y compounds disclosed herein are commercially available, are known in the scientific literature, or can be made using procedures familiar to the person of ordinary skill in the art, provided from commercial sources, or the general procedures described in the Examples below. [00238] For example, in various embodiments, H-A6-A7-Y is selected from any one of [00239] In various embodiments as described herein, H-A5-A6-A7-Y is selected from the are as otherwise described herein. The various H-A5-A6-A7-Y compounds disclosed herein are commercially available, are known in the scientific literature, or can be made using procedures familiar to the person of ordinary skill in the art, provided from commercial sources, or the general procedures described in the Examples below. [00240] In various embodiments as described herein, HOOC-A6-A7-Y is HOOC-A6-A7-Y compounds disclosed herein are commercially available, are known in the scientific literature, or can be made using procedures familiar to the person of ordinary skill in the art, provided from commercial sources, or the general procedures described in the Examples below. [00241] Many general references providing commonly known chemical synthetic schemes and conditions useful for synthesizing materials/intermediates used in the synthesis of the disclosed compounds are available (see, e.g., Smith, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Eighth Edition, Wiley- Interscience, 2019; or Furniss, Hannaford, Smith, Tatchelll, Vogel’s A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fifth Edition, New York: Longman, 1989). [00242] Compounds as described herein can be purified by any of the means known in the art, including chromatographic means, such as HPLC, preparative thin layer chromatography, flash column chromatography and ion exchange chromatography. Any suitable stationary phase can be used, including normal and reversed phases as well as ionic resins. Most typically, the disclosed compounds are purified via silica gel and/or alumina chromatography. See, e.g., Still, Kahn, Mitra, J. Org. Chem.1978, 43, 2923-292, Introduction to Modern Liquid Chromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, New York, 1969. [00243] During any of the processes for preparation of the subject compounds, it can be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This can be achieved by means of conventional protecting groups as described in standard works, such as J. F. W. McOmie, "Protective Groups in Organic Chemistry,” Plenum Press, London and New York 1973, in P. G. M. Wuts, " Greene’s Protective Groups in Organic Synthesis,” Firth edition, Wiley, New York 2014, in "The Peptides"; Volume 3 (editors: E. Gross and J. Meienhofer), Academic Press, London and New York 1981, in "Methoden der organischen Chemie,” Houben-Weyl, 4.sup.th edition, Vol.15/l, Georg Thieme Verlag, Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, "Aminosauren, Peptide, Proteine,” Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982, and/or in Jochen Lehmann, "Chemie der Kohlenhydrate: Monosaccharide and Derivate,” Georg Thieme Verlag, Stuttgart 1974. The protecting groups can be removed at a convenient subsequent stage using methods known from the art. [00244] The compounds disclosed herein can be made using procedures familiar to the person of ordinary skill in the art and as described herein. For example, compounds of structural formulas M5 and M4 can be prepared according to Schemes 1, 2, 3, 4, general procedures (see the Examples below), and/or analogous synthetic procedures. One of skill in the art can adapt the reaction sequences of Schemes 1, 2, 3, and 4, general procedures, and Examples described to fit the desired target molecule. Of course, in certain situations one of skill in the art will use different reagents to affect one or more of the individual steps or to use protected versions of certain of the substituents. Additionally, one skilled in the art would recognize that compounds of the disclosure can be synthesized using different routes altogether. Lipid Nanoparticles (LNPs) and Targeted LNPs (tLNPs) [00245] In certain aspects, this disclosure provides an LNP comprising an ionizable cationic lipid of formula M4 and/or M5. In some embodiments, an LNP comprises an ionizable cationic lipid of formula M4 and/or M5 and a phospholipid, a sterol, a co-lipid, a PEGylated lipid, or a combination thereof. In certain embodiments, the PEG-lipids are not functionalized PEG-lipids. In other embodiments, the PEG-lipids are functionalized PEG- Lipids. In certain embodiments, the LNP comprises at least one PEG-lipid that is functionalized and at least one PEG-lipid that is not functionalized. [00246] In further aspects, this disclosure provides a targeted lipid nanoparticle (tLNP) comprising an ionizable cationic lipid of formula M4 and/or M5. In some embodiments, the aforementioned tLNP can further comprise one or more of a phospholipid, a sterol, a co-lipid, and a PEG-lipid, or a combination thereof, and a functionalized PEG-lipid. As used herein, “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid. The functionalized PEG-lipid can be reacted with a binding moiety (e.g., an antibody or Fab) after the LNP is formed, so that the binding moiety is conjugated to the PEG portion of the lipid. The conjugated binding moiety can thus serve as a targeting moiety for the tLNP. [00247] In various embodiments, a binding moiety of a LNP or tLNP comprises an antigen binding domain, an antigen, a ligand-binding domain of a receptor, or a receptor ligand. In some embodiments, a binding moiety comprises a complete antibody, an F(ab)2, an Fab, a minibody, a single-chain Fv (scFv), a diabody, a VH domain, or a nanobody, such as a VHH or single domain antibody. In some embodiments, the receptor ligand is a carbohydrate, for example, a carbohydrate comprising terminal galactose or N-acetylgalactosamine units, which are bound by the asialoglycoprotein receptor. These binding moieties constitute means for LNP targeting. Some embodiments specifically include one or more of these binding moieties. Other embodiments specifically exclude one or more of these binding moieties. LNP and tLNP Compositions [00248] The LNP composition contributes to the formation of stable LNPs and tLNPs, efficient encapsulation of a payload, protection of a payload from degradation until it is delivered into a cell, and promotion of endosomal escape of a payload into the cytoplasm. These functions are primarily independent of the specificity of the binding moiety (or moieties) serving to direct or bias a tLNP to a particular cell type(s). Additional LNP and tLNP compositions are generally disclosed in PCT/US2024/032141, filed 31 May 2024, published as WO2024/249954, and entitled Lipid Nanoparticle Formulations and Compositions, which is incorporated by reference for all that it teaches about the design, formation, characterization, properties, and use of LNPs and tLNPs. [00249] The LNPs and/or tLNPs can include the various components in amounts sufficient to provide a nanoparticle with a desired shape, fluidity, and bio-acceptability as described herein. With respect to LNPs or tLNPs of this disclosure, in some embodiments, the LNPs (or tLNP) comprises at least one ionizable cationic lipid as described herein in an amount in the range of from about 35 to about 65 mol%, e.g., in an amount of from about 40 to about 65 mol%, about 40 to about 60 mol%. In some embodiments, the LNP or tLNP comprises about 58 mol% or 62 mol% ionizable cationic lipid. In some embodiments, is the LNP (or tLNP) comprises a phospholipid in an amount in the range of from about 7 to about 30 mol%, e.g., in an amount of from about 13 to about 30 mol%. In some embodiments, the LNP or tLNP comprises about 10 mol% phospholipid. In some embodiments, the LNP (or tLNP) comprises a sterol in an amount in the range of from about 20 to about 50 mol%, e.g., in an amount in the rage of from about 20 to about 45 mol%, or about 30 to about 50 mol%, or about 30 to about 45 mol%. In some embodiments, the LNP or tLNP comprises about 30.5, 26.5, or 23.5 mol% sterol. In some embodiments, the LNP (or tLNP) comprises at least one co-lipid in an amount in the range of from about 1 to about 30 mol%. In some embodiments, the LNP (or tLNP) comprises at least one unfunctionalized PEG-lipid in an amount of from 0 to about 5 mol%, e.g., in the range of amount 0 to about 3 mol%, or about 0.1 to about 5 mol%, or about 0.5 to about 5 mol%, or about 0.5 to about 3 mol%. In some embodiments, the LNP or tLNP comprises about 1.4 mol% unfunctionalized PEG-lipid. In some embodiments, the LNP or tLNP comprises at least one functionalized PEG-lipid in an amount in the range of from about 0.1 to about 5 mol%, e.g., in the range of from about 0.1 to 0.3 mol%. In some embodiments, the LNP or tLNP comprises about 0.1 mol% functionalized PEG-lipid. In some embodiments, the functionalized PEG-lipid is conjugated to a binding moiety. [00250] In certain aspects, this disclosure provides an LNP or tLNP, wherein the LNP or tLNP comprises about 35 mol% to about 65 mol% of an ionizable cationic lipid, about 0.5 mol% to about 3 mol% of a PEG-lipid (including non-functionalized PEG-lipid and optionally a functionalized PEG-lipid), about 7 mol% to about 13 mol% of a phospholipid, and about 30 mol% to about 50 mol% of a sterol. In some embodiments, an LNP or tLNP comprises a payload with a net negative charge for example, a peptide, a polypeptide, a protein, a small molecule, or a nucleic acid molecule, and combinations thereof. A payload is generally encompassed by or in the interior of an LNP or tLNP. As disclosed herein dosages always refer to the amount of payload being provided. In some embodiments, a payload comprises one or more species of nucleic acid molecule. For tLNP encapsulating mRNA dosages are typically in the range of 0.05 to 5 mg/kg without regard for recipient species. In some embodiments, the dosage is in the range of 0.1 to 1 mg/kg. [00251] With respect to LNPs or tLNPs of this disclosure, in some embodiments, the ratio of total lipid to nucleic acid is about 10:1 to about 50:1 on a weight basis. In some embodiments, the ratio of total lipid to nucleic acid is about 10:1, about 20:1, about 30:1, or about 40:1 to about 50:1, or 10:1 to 20:1, 30:1, 40:1 or 50:1, or any range bound by a pair of these ratios. The ratio of lipid to nucleic acid can also be reported as an N/P ratio, the ratio of positively chargeable lipid amine (N = nitrogen) groups to negatively-charged nucleic acid molecule phosphate (P) groups. In some instances, the N/P ratio is from about 3 to about 9, about 3 to about 7, about 3 to about 6, about 4 to about 6, about 5 to about 6, or about 6. In some instances, the N/P ratio is from 3 to 9, 3 to 7, 3 to 6, 4 to 6, 5 to 6, or 6. In some embodiments, an LNP or tLNP comprises about 40 mol% to about 62 mol% ionizable cationic lipid. In some embodiments, an LNP or tLNP comprises about 1 mol% to about 2 mol% total PEG-lipid. In certain embodiments, an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol%, for example about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% functionalized PEG-lipid. In some embodiments, a binding moiety is conjugated to functionalized PEG-lipid. In certain instances, a tLNP is an LNP that further comprises an antibody (for example, a whole IgG) as the binding moiety which is present at an antibody:mRNA ratio (w/w) of about 0.3 to about 1.0 w/w. [00252] Due to physiologic and manufacturing constraints LNP or tLNP, particles with a hydrodynamic diameter of about 50 to about 150 nm are desirable for in vivo use. Accordingly, in some embodiments, the LNP or tLNP has a hydrodynamic diameter of 50 to 150 nm and in some embodiments the hydrodynamic diameter is ≤120, ≤110, ≤100, or ≤90 nm. Uniformity of particle size is also desirable with a polydispersity index (PDI) of ≤0.2 (on a scale of 0 to 1) being acceptable. Both hydrodynamic diameter and polydispersity index are determined by dynamic light scattering (DLS). Particle diameter as assessed from cryo- transmission electron microscopy (Cryo-TEM) can be smaller than the DLS-determined value. Phospholipids [00253] As described above, in various embodiments, the LNPs and tLNPs include a phospholipid. As would be understood by the person or ordinary skill in the art, phospholipids are amphiphilic molecules. Due to the amphiphilic nature of phospholipids, these molecules are known to form bilayers and by including them in the LNPs and tLNPs, as described herein, they can provide membrane formation, stability, and rigidity. As used herein, phospholipids include a hydrophilic head group, including a functionalized phosphate group, and two hydrophobic tail groups derived from fatty acids. For example, in various embodiments as described herein, the phospholipids include a phosphate group functionalized with ethanolamine, choline, glycerol, serine, or inositol. As described above, the phospholipid includes two hydrophobic tail groups derived from fatty acids. These hydrophobic tail groups can be derived from unsaturated or saturated fatty acids. For example, the hydrophobic tail groups can be derived from a C12-C20 fatty acid. [00254] With respect to LNPs or tLNPs of this disclosure, in various embodiments, a phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3- phosphocholine (DAPC), or a combination thereof. In various embodiments, the phospholipid is selected from the group comprising dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2- diarachidoyl-sn-glycero-3-phosphocholine (DAPC). In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC). Phospholipids can contribute to formation of a membrane, whether monolayer, bilayer, or multi-layer, surrounding the core of the LNP or tLNP. Additionally, phospholipids such as DSPC, DMPC, DPPC, DAPC impart stability and rigidity to membrane structure. Phospholipids, such as DOPE, impart fusogenicity. Further phospholipids, such as DMPG, which attains negative charge at physiologic pH, facilitates charge modulation. Thus, phospholipids constitute means for facilitating membrane formation, means for imparting membrane stability and rigidity, means for imparting fusogenicity, and means for charge modulation. [00255] In some embodiments, an LNP or tLNP has about 7 mol% to about 13 mol% phospholipid, about 7 mol% to about 10 mol% phospholipid, or about 10 mol% to about 13 mol% phospholipid. In certain embodiments, an LNP has about 7 mol%, about 10 mol%, or about 13 mol% phospholipid. In certain instances, the phospholipid is DSPC. In certain instances, the phospholipid is DAPC. Sterols [00256] The disclosed LNP and tLNP comprise a sterol. Sterol refers to a subgroup of steroids that contain at least one hydroxyl (OH) group. More specifically, a gonane derivative with an OH group substituted for an H at position 3, or said differently, but equivalently, a steroid with an OH group substituted for an H at position 3. Examples of sterols include, without limitation, cholesterol, ergosterol, β-sitosterol, stigmasterol, stigmastanol, 20- hydroxycholesterol, 22-hydroxycholesterol, and the like. With respect to LNPs or tLNPs of this disclosure, in various embodiments, a sterol is cholesterol, 20-hydroxycholesterol, 22- hydroxycholesterol, or a phytosterol. In further embodiments the phytosterol comprises campesterol, sitosterol, or stigmasterol, or combinations thereof. In preferred embodiments, the cholesterol is not animal-sourced but is obtained by synthesis using a plant sterol as a starting point. LNPs incorporating C-24 alkyl (such as methyl or ethyl) phytosterols have been reported to provide enhanced gene transfection. The length of the alkyl tail, the flexibility of the sterol ring, and polarity related to a retain C-3 -OH group are important to obtaining high transfection efficiency. While β-sitosterol and stigmasterol performed well, vitamin D2, D3 and calcipotriol, (analogs lacking intact body of cholesterol) and betulin, lupeol ursolic acid and olenolic acid (comprising a 5th ring) should be avoided. Sterols serve to fill space between other lipids in the LNP or tLNP and influence LNP or tLNP shape. Sterols also control fluidity of lipid compositions, reducing temperature dependence. Thus, sterols such as cholesterol, 20-hydroxycholesterol, 22-hydroxycholesterol, campesterol, fucosterol, β-sitosterol, and stigmasterol constitute means for controlling LNP shape and fluidity or sterol means for increasing transfection efficiency. In designing a lipid composition for a LNP or tLNP, in some embodiments, sterol content can be chosen to compensate for different amounts of other types of lipids, for example, ionizable cationic lipid or phospholipid. [00257] In some embodiments, an LNP or tLNP has about 27 mol% or about 30 mol% to about 50 mol% sterol, or about 30 mol% to about 38 mol% sterol. In certain embodiments, an LNP or tLNP has about 30.5 mol%, about 33.5 mol%, or about 37.5 mol% sterol. In certain instances, the sterol is cholesterol. In certain embodiments, the sterol is a mixture of sterols, for example, cholesterol and β-sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, the sterol is about 25 mol% 20-hydroxycholesterol and about 75 mol% cholesterol. In some instances, the sterol is about 25 mol% β-sitosterol and about 75 mol% cholesterol. In some instances, the sterol is about 50 mol% β-sitosterol and about 50 mol% cholesterol. In certain embodiments, an LNP or tLNP has 27 mol% or 30 mol% to 50 mol% sterol or 30 mol% to 38 mol% sterol. In further embodiments, an LNP or tLNP has 30.5 mol%, 33.5 mol%, or 37.5 mol% sterol. In certain instances, a sterol is cholesterol. In certain embodiments, a sterol is a mixture of sterols, for example, cholesterol and β-sitosterol or cholesterol and 20-hydroxycholesterol. In some instances, a sterol is 25 mol% 20- hydroxycholesterol and 75 mol% cholesterol. In further instances, a sterol is 25 mol% β- sitosterol and 75 mol% cholesterol. In still further instances, a sterol is 50 mol% β-sitosterol and 50 mol% cholesterol. [00258] With respect to LNPs or tLNPs of this disclosure, in some embodiments, a co- lipid is absent or comprises an ionizable lipid, anionic or cationic. A co-lipid can be used to adjust various properties of an LNP or tLNP, such as surface charge, fluidity, rigidity, size, stability, and the like properties. In some embodiments, a co-lipid is an ionizable lipid, such as cholesterol hemisuccinate (CHEMS) or an ionizable lipid of this disclosure. In some embodiments, a co-lipid is a charged lipid, such as a quaternary ammonium head group containing lipid. In some embodiments, a quaternary ammonium head group containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3β-(N-(N’,N’- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof. In certain embodiments, these compounds a chloride, bromide, mesylate, or tosylate salt. As described above, when quaternary ammonium head group containing lipids are included in LNPs or tLNPs, fatal hyperacute toxicity in laboratory animals has been observed. Accordingly, when the co-lipid is a quaternary ammonium head group containing lipid, the quaternary ammonium head group containing lipid is present it makes up no more than 50 mol% of the total cationic lipid, for example, from 5 to 50% of the total cationic lipid. For illustration, if an LNP or tLNP were to have cationic lipid content of 70 mol% and 5 to 50 mol% of the total cationic lipid as quaternary ammonium lipid, the LNP or tLNP would have from 3.5 mol% quaternary ammonium lipid and 66.5 mol% ionizable cationic lipid to 35 mol% each of quaternary ammonium lipid and ionizable cationic lipid. [00259] When the disclosed ionizable lipids of formulas M4 and M5 have a measured pKa ranging from about 6 to about 7 or from 6 to 7, they can contribute substantial endosomal release activity to an LNP or tLNP containing the ionizable lipid. More acidic or basic ionizable lipids of formulas M4 and M5 can contribute surface charge and thus serve as a co-lipid as described immediately above. In such cases, it can be advantageous to incorporate another lipid with fusogenic activity into an LNP or tLNP of this disclosure. Surface charge is known to influence the tissue tropism of LNPs or tLNPs; for example, positively charged LNPs or tLNPs have shown a tropism for spleen and lung. PEG-Lipids [00260] With respect to a LNP or tLNP of this disclosure, a PEG-lipid is a lipid conjugated to a polyethylene glycol (PEG). In some embodiments as described herein, the PEG-lipid is a C14-C20 lipid conjugated with a PEG. For example, in various embodiments as described herein, the PEG-lipid is a C14-C20 lipid conjugated with a PEG, or a C14-C18 lipid conjugated with a PEG, or a C14-C16 lipid conjugated with a PEG. In certain embodiments as described herein, the PEG-lipid is a fatty acid conjugated with a PEG. The fatty acid of the PEG-lipid can have a variety of chain lengths. For each, in some embodiments, the PEG-lipid is a fatty acid conjugated with PEG, wherein the fatty acid chain length is in the range of C14-C20 (e.g., in the range of C14-C18, or C14-C16). PEG-lipids with fatty acid chain lengths less than C14 are too rapidly lost from the (t)LNP while those with chain lengths greater than C20 are prone to difficulties with formulation. [00261] In certain aspects, the LNP comprises one or more PEG-lipids and/or functionalized PEG-lipids; when both a functionalized and unfunctionalized PEG-lipid, the PEG-lipid present they can be the same or different; and one or more ionizable cationic lipids; the LNP can further comprise a phospholipid, a sterol, a co-lipid, or any combination thereof. The term “functionalized PEG-lipid” refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group that can be used for conjugating a targeting moiety to the PEG-lipid. The functionalized PEG-lipid can be reacted with a binding moiety so that the binding moiety is conjugated to the PEG portion of the lipid. The conjugated binding moiety can thus serve as a targeting moiety for the LNP to constitute a tLNP. In some embodiments, the binding moiety is conjugated to the functionalized PEG- lipid after an LNP comprising the functionalized PEG-lipid is formed. In other embodiments, the binding moiety is conjugated to the PEG-lipid and then the conjugate is inserted into a previously formed LNP. [00262] In certain embodiments, the LNP is a tLNP comprising one or more functionalized PEG-lipids that has been conjugated to a binding moiety. In certain embodiments, the tLNP also comprises PEG-lipids not functionalized or conjugated with a binding moiety. In some embodiments, the functionalization is a maleimide. In some embodiments the functionalization is a bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide moiety at the terminal hydroxyl end of the PEG moiety. In some embodiments, the binding moiety comprises an antibody or antigen binding portion thereof. In some embodiments, the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group. In some embodiments, the conjugation linkage comprises a reaction product of a thiol in the binding moiety with a functionalized PEG-lipid. In some embodiments, the functionalization is a maleimide, azide, alkyne, dibenzocyclooctyne (DBCO), bromomaleimide or bromomaleimide amide, alkynylamide, or alkynylimide. In some embodiments, the binding moiety comprises an antibody or antigen binding portion thereof. In some embodiments, the binding moiety is a polypeptide comprising a binding domain and an N- or C-terminal extension comprising an accessible thiol group. [00263] PEG can be made in a large range of sizes. In certain embodiments, the PEG of the disclosed LNP and tLNP is PEG-1000 to PEG-5000. It is to be understood that polyethylene preparations of these sizes are polydisperse and that the nominal size indicates an approximate average molecular weight of the distribution. Taking the molecular weight of an individual repeating unit of (OCH2CH2)n to be 44, a PEG molecule with n=22 would have a molecular weight of 986, with n=45 a molecular weight of 1998, and with n=113 a molecular weight of 4990. n≈22 to 113 is used to represent PEG-lipids incorporating PEG moieties in the range of PEG-1000 to PEG-5000 such as PEG-1000, PEG-1500, PEG- 2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000, although some molecules from preparations at the average molecular weight boundaries will have an n outside that range. For individual preparations n≈22 is used to represent PEG-lipids incorporating PEG moieties from PEG-1000, n≈45 is used to represent PEG-lipids incorporating PEG moieties from PEG-2000 n≈67 is used to represent PEG-lipids incorporating PEG moieties from PEG-3000, n≈90 is used to represent PEG-lipids incorporating PEG moieties from PEG-4000, n≈113 is used to represent PEG-lipids incorporating PEG moieties from PEG-5000. Some embodiments incorporate PEG moieties in a range bounded by any pair of the foregoing values of n or average molecular weight. In some embodiments of the PEG-lipid, a PEG is of 500-5000 or 1000-5000 Da molecular weight (MW). For example, in some embodiments, the PEG of the PEG-lipid has a molecular weight in the range of 1500-5000 Da or 2000-5000 Da. In some embodiments as described herein, the PEG-lipid has a molecular weight in the range of 500-4000 Da, or 500-3000 Da, or 1000-4000 Da, or 1000-3000, or 1000-2500, or 1500-4000, or 1500-3000, or 1500-2500 Da. In some embodiments, the PEG unit has a MW of 2000 Da (sometime abbreviated as PEG(2k)). Some embodiments incorporate PEG moieties of PEG-1000, PEG-2000, or PEG- 5000. Certain embodiments comprise a DSG-PEG, for example, DSG-PEG-2000. Certain embodiments comprise a DSPE-PEG, for example, DSPE-PEG-2000. Certain embodiments comprise both DSG-PEG-2000 and/or DSPE-PEG2000. [00264] In some embodiments, the PEG moiety is PEG-500 to PEG-5000 such as PEG- 500, PEG-1000, PEG-1500, PEG-2000, PEG-2500, PEG-3000, PEG-3500, PEG-4000, PEG-4500, and PEG-5000. In some instances, the PEG moiety is PEG-2000. In some embodiments, the PEG unit has a MW of 2000 Da. [00265] Common PEG-lipids fall into two classes diacyl glycerols and diacyl phospholipids. Examples of diacyl glycerol PEG-lipids include DMG-PEG (1,2-dimyristoyl- glycero-3-methoxypolyethylene glycol), DPG-PEG (1,2-dipalmitoyl-glycero-3- methoxypolyethylene glycol), DSG-PEG (1,2-distearoyl-glycero-3-methoxypolyethylene glycol), and DOG-PEG (1,2-dioleoyl-glycero-3-methoxypolyethylene glycol). Examples of diacyl phospholipids include DMPE-PEG (1,2-dimyristoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol), DPPE-PEG (1,2-dipalmitoyl-glycero-3-phosphoethanolamine- 3-methoxypolyethylene glycol), DSPE-PEG (1,2-distearoyl-glycero-3-phosphoethanolamine- 3-methoxypolyethylene glycol), and DOPE-PEG (1,2-dioleoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol). [00266] In some embodiments, the MW2000 PEG-lipid (e.g., a PEG-lipid comprising a PEG of a molecular weight of 2000 Da) comprises DMG-PEG2000 (1,2-dimyristoyl-glycero- 3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3- methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl-glycero-3- methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE- PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol- 2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), or combinations thereof. In some embodiments, the PEG unit has a MW of 2000 Da. In some embodiments, the MW2000 PEG-lipid comprises DMrG- PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DPrG-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSrG-PEG2000 (1,2- distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DorG-PEG2000 (1,2-dioleoyl- glycero-3-methoxypolyethylene-rac-glycol-2000), DMPEr-PEG200 (1,2-dimyristoyl-rac- glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPEr-PEG2000 (1,2-dipalmitoyl-rac-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPEr-PEG2000 (1,2-distearoyl-rac-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), DOPEr-PEG2000 (1,2-dioleoyl-rac-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), or combinations thereof. The glycerol in these lipids is chiral. Thus, in some embodiments, the PEG-lipid is racemic. Alternatively, optically pure antipodes of the glycerol portion can be employed, that is, the glycerol portion is homochiral. As used herein with respect to glycerol moieties, optically pure means ≥95% of a single enantiomer (D or L). In some embodiments, the enantiomeric excess is ≥98%. In some embodiments, the enantiomeric excess is ≥99%. Additional PEG- lipids, including achiral PEG-lipids built on a symmetric dihydroxyacetone scaffold, a symmetric 2-(hydroxymethyl)butane-1,4-diol, or a symmetric glycerol scaffold, are disclosed in U.S. Provisional Application No.63/362,502, filed on April 5, 2022, and PCT/US2023/017648 filed on April 5, 2023 (WO 2023/196445), both entitled PEG-Lipids and Lipid Nanoparticles, which are incorporated by reference in their entirety. [00267] The above examples are presented as methoxypolyethylene glycols, but the terminus need not necessarily be methoxyl. With respect to any of the PEG-lipids that have not been functionalized, in alternative embodiments, the PEG moiety of the PEG lipids can terminate with a methoxyl, a benzyloxyl, a 4-methoxybenzyloxyl, or a hydroxyl group (that is, an alcohol). The terminal hydroxyl facilitates functionalization. The methoxyl, benzyloxyl, and 4-methoxybenzyloxyl groups are advantageously provided for PEG-lipid that will be used without functionalization as a component of the LNP. However, all four of these alternatives are useful as the (non-functionalized) PEG-lipid component of LNPs. The 4- methoxybenzyloxyl group, often used as a protecting group during synthesis of the PEG- lipid, is readily removed to generate the corresponding hydroxyl group. Thus, the 4- methoxybenzyloxyl group offers a convenient path to the alcohol when it is not synthesized directly. The alcohol is useful for being functionalized, prior to incorporation of the PEG-lipid into a LNP, so that a binding moiety can be conjugated to it as a targeting moiety for the LNP (making it a tLNP). As used herein, the terminus of the PEG moiety, and similar constructions, refers to the end of the PEG moiety that is not attached to the lipid. [00268] A PEG-moiety provides a hydrophilic surface on the LNP, inhibiting aggregation or merging of LNP, thus contributing to their stability and reducing polydispersity, i.e. reducing the heterogeneity of a dispersion of LNPs. Additionally, a PEG moiety can impede binding by the LNP, including binding to plasma proteins. These plasma proteins include apoE which is understood to mediate uptake of LNP by the liver so that inhibition of binding can lead to an increase in the proportion of LNP reaching other tissues. These plasma proteins also include opsonins so that inhibition of binding reduces recognition by the reticuloendothelial system. The PEG-moiety can also be functionalized to serve as an attachment point for a targeting moiety. Conjugating a cell- or tissue-specific binding moiety to the PEG-moiety enables a tLNP to avoid the liver and bind to its target tissue or cell type, greatly increasing the proportion of LNP that reaches the targeted tissue or cell type. PEG- lipid can thus serve as means for inhibiting LNP binding, and PEG-lipid conjugated to a binding moiety can serve as means for LNP-targeting. [00269] As used herein, the term “functionalized PEG-lipid” and similar constructions refer generally to both the unreacted and reacted entities. The lipid composition of a LNP can be described referencing the reactive species even after conjugation has taken place (forming a tLNP). For example, a lipid composition can be described as comprising DSPE-PEG- maleimide and can be said to further comprise a binding moiety without explicitly noting that upon reaction to form the conjugate the maleimide will have been converted to a succinimide (or hydrolyzed succinimide). Similarly, if the reactive group is bromomaleimide, after conjugation it will be maleimide. These differences of chemical nomenclature for the unreacted and reacted species are to be implicitly understood even when not explicitly stated. Certain embodiments comprise a DSG-PEG, for example, DSG-PEG-2000. Certain embodiments comprise a functionalized DSPE-PEG, for example, functionalized DSPE- PEG-2000. Certain embodiments comprise both DSG-PEG-2000 and functionalized DSPE- PEG-2000. In some instances, the functionalized PEG-lipid is functionalized with a maleimide moiety, for example, DSPE-PEG-2000-MAL. Analogously, in certain embodiments, DSG-PEG serves as the functionalized or functionalized and non- functionalized PEG-lipid. [00270] In certain embodiments, the PEG-lipid and/or functionalized PEG-lipid comprises a scaffold selected from Formula S1, Formula S2, Formula S3, or Formula S4: Formula S1 Formula S2 Formula S3 Formula S4 wherein represents the points of ester connection with a fatty acid, and represents the point of ester (S1) or ether (S2, S3, and S4) formation with the PEG moiety. In some embodiments, the fatty acid esters are C14-C20 straight-chain alkyl fatty acids. In some embodiments, the PEG moiety is functionalized and the fatty acid esters are C16-C20 straight- chain alkyl fatty acids. For example, the straight-chain alkyl fatty acid is C14, C15, C16, C17, C18, C19, or C20. In some embodiments, the fatty acid esters are C14-C20 symmetric branched-chain alkyl fatty acids. For example, the branched-chain alkyl fatty acid is C14, C15, C16, C17, C18, C19, or C20. By symmetric it is meant that each alkyl branch has the same number of carbons. In some embodiments, the branch is at the 3, 4, 5, 6, or 7 position of the fatty acid ester. The synthesis and use of PEG-lipids built on scaffolds S1-S4 is disclosed in WO2023/196445A1 which is incorporated by reference for all that it teaches about PEG-lipids and their use. [00271] Some embodiments of the disclosed ionizable cationic lipids have head groups with small (<250 Da) PEG moieties. These lipids are not what is meant by the term PEG- lipid as used herein. These small PEG moieties are generally too small to impede binding to a similar extent as the larger PEG moieties of the PEG-lipids disclosed above, though they will impact the lipophilicity of ionizable cationic lipid. Moreover, the PEG-lipids are understood to be primarily located in an exterior facing lamella whereas much of the ionizable cationic lipid is in the interior of the LNP. [00272] In certain embodiments, a functionalized PEG-lipid of a LNP or tLNP of this disclosure comprises one or more fatty acid tails, each of which is no shorter than C16 nor longer than C20 for straight-chain fatty acids. For branched chain fatty acids, tails no shorter than C14 fatty acids nor longer than C20 are acceptable. In some embodiments, fatty acid tails are C16. In some embodiments, the fatty acid tails are C18. In some embodiments, the functionalized PEG-lipid comprises a dipalmitoyl lipid. In some embodiments, the functionalized PEG-lipid comprises a distearoyl lipid. The fatty acid tails serve as means to anchor the PEG-lipid in the tLNP to reduce or eliminate shedding of the PEG-lipid from the tLNP. This is a useful property for the PEG-lipid whether or not it is functionalized but has greater significance for the functionalized PEG-lipid as it will have a targeting moiety attached to it and the targeting function could be impaired if the PEG-lipid (with the conjugated binding moiety, such as an antibody) were shed from the tLNP. [00273] In some embodiments, an LNP or tLNP comprises about 0.5 mol% to about 3 mol% or 0.5 mol% to 3 mol% PEG-lipid comprising functionalized and non-functionalized PEG-lipid. In certain embodiments, an LNP or tLNP comprises DSG-PEG. In other embodiments, an LNP or tLNP comprises DMG-PEG or DPG-PEG. In certain embodiments, an LNP or tLNP comprises DSPE-PEG. In some embodiments, the functionalized and non- functionalized PEG-lipids are not the same PEG-lipid, for example, the non-functionalized PEG-lipid can be a diacylglycerol and the functionalized PEG-lipid a diacyl phospholipid. tLNP with such mixtures have reduced expression in the liver, possibly due to reduced uptake. In certain embodiments the functionalized PEG-lipid is DSPE-PEG and the non- functionalized PEG-lipid is DSG-PEG. In some embodiments, an LNP or tLNP comprises about 0.4 mol% to about 2.9 mol% or about 0.9 mol% to about 1.4 mol% non-functionalized PEG lipid. In certain embodiments, an LNP or tLNP comprises about 1.4 mol% or 1.4 mol% non-functionalized PEG lipid. In some embodiments, an LNP or tLNP comprises about 0.1 mol% to about 0.3 mol% or 0.1 mol% to 0.3 mol% functionalized lipid. In some instances, the functionalized lipid is DSPE-PEG. In certain instances, an LNP or tLNP comprises about 0.1 mol%, about 0.2 mol%, or about 0.3 mol% DSPE-PEG. In certain instances, an LNP or tLNP comprises 0.1 mol%, 0.2 mol%, or 0.3 mol% DSPE-PEG. In certain instances, the functionalized PEG-lipid is conjugated to a binding moiety. As used herein, the phrase “is conjugated to” and similar constructions are meant to convey a state of being, that is, a structure, and not a process, unless context dictates otherwise. Conjugation [00274] Any suitable chemistry can be used to conjugate the binding moiety to the PEG of the PEG-lipid, including maleimide (see Parhiz et al., J. Controlled Release 291:106, 2018) and click (see Kolb et al., Angewandte Chemie International Edition 40(11):2004, 2001; and Evans, Australian J. Chem.60(6):384, 2007) chemistries. Reagents for such reactions include lipid-PEG-maleimide, lipid-PEG-cysteine, lipid-PEG-alkyne, lipid, PEG- dibenzocyclooctyne (DBCO), and lipid-PEG-azide. Further conjugations reactions make use of lipid-PEG-bromo maleimide, lipid-PEG-alkylnoic amide, PEG-alkynoic imide, and lipid- PEG-alkyne reactions, as disclosed in U.S. Provisional Application No.63/362,502, filed on April 5, 2022, and PCT/US2023/017648 filed on April 5, 2023 (WO 2023/196445), both entitled PEG-Lipids and Lipid Nanoparticles, which are incorporated by reference in their entirety. On the binding moiety side of the reaction can be used an existing cysteine sulfhydryl, or the protein derivatized by adding a sulfur containing carboxylic acid, for example, to the epsilon amino of a lysine to react with a maleimide, bromomaleimide, alkylnoic amide, or alkynoic imide. In certain embodiments, to modify an epsilon amino of a binding moiety lysine to react with a maleimide functionalized PEG-lipid, the binding moiety (e.g., an antibody) can be reacted with N-succinimidyl S-acetylthioacetate (SATA). SATA is then deprotected, for example, using 0.5 M hydroxylamine followed by removal of the unreacted components by G-25 Sephadex Quick Spin Protein columns (Roche Applied Science, Indianapolis, IN). The reactive sulfhydryl group on the binding moiety is then conjugated to maleimide moieties on LNPs of the disclosure using thioether conjugation chemistry. Alternatively, an alkyne can be added to a sulfhydryl or an epsilon amino of a lysine to participate in a click chemistry reaction. [00275] Purification can be performed using Sepharose CL-4B gel filtration columns (Sigma-Aldrich). tLNPs (LNPs conjugated with a targeting antibody) can be stored frozen at - 80°C until needed. Others have conjugated antibody to free functionalized PEG-lipid and then incorporated the conjugated lipid into pre-formed LNP. However, it was found that this procedure is more controllable and produces more consistent results. [00276] There are also several approaches to site-specific conjugation. Particularly but not exclusively suitable for truncated forms of antibody, C-terminal extensions of native or artificial sequences containing a particularly accessible cysteine residue are commonly used. Partial reduction of cysteine bonds in an antibody, for example, with tris(2- carboxy)phosphine (TCEP), can also generate thiol groups for conjugation which can be site-specific under defined conditions with an amenable antibody fragment. Alternatively, the C-terminal extension can contain a sortase A substrate sequence, LPXTG (SEQ ID NO: 1) which can then be functionalized in a reaction catalyzed by sortase A and conjugated to the PEG-lipid, including through click chemistry reactions (see, for example, Moliner-Morro et al., Biomolecules 10(12):1661, 2020 which is incorporated by reference herein for all that it teaches about antibody conjugations mediated by the sortase A reaction and/or click chemistry). The use of click chemistry for the conjugation of a targeting moiety, such as various forms of antibody, is disclosed, for example, in WO2024/102,770 which is incorporated by reference in its entirety for all that it teaches about the conjugation of targeting moieties to LNPs that is not inconsistent with this disclosure. [00277] For whole antibody and other forms comprising an Fc region, site-specific conjugation to either (or both) of two specific lysine residues (Lys248 and Lys288) can be accomplished without any change to or extension of the native antibody sequence by use of one of the AJICAP® reagents (see, for example, Matsuda et al., Mol. Pharmaceutics 18:4058, 2021 and Fujii et al., Bioconjugate Chem.34:728, 2023, which are incorporated by reference herein for all that they teach regarding conjugation of antibodies with AJICAP reagents). AJICAP reagents are modified affinity peptides that bind to specific loci on the Fc and react with an adjacent lysine residue to form an affinity peptide conjugate of the antibody. The peptide is then cleaved with base to leave behind a thiol-functionalized lysine residue which can then undergo conjugation through maleimide or haloamide reactions, for example). Functionalization with azide or dibenzocyclooctyne (DBCO) for conjugation by click chemistry is also possible. This and similar technology are further described in US 2020/0190165 (corresponding to WO 2018/199337), US 2021/0139549 (corresponding to WO 2019/240287) and US 2023/0248842 (corresponding to WO 2020/184944) which are incorporated by reference in their entirety for all that they teach about such modified affinity peptides and their use. [00278] Accordingly, in some embodiments the binding moiety is conjugated to the PEG moiety of the PEG-lipid through a thiol modified lysine residue. In some embodiments, the conjugation is through a cysteine residue in a native or added antibody sequence. In other embodiments, the conjugation is through a sortase A substrate sequence. In still other embodiments, the conjugation is through a specific lysine residue (Lys248 or Lys288) in the Fc region. Binding Moieties [00279] The tLNP of the various disclosed aspects comprise a binding moiety, such as an antibody or antigen binding domain thereof or a cell surface receptor ligand. As used herein, a “binding moiety” or “targeting moiety” refers to a protein, polypeptide, oligopeptide or peptide, carbohydrate, nucleic acid, or combinations thereof capable of specifically binding to a target or multiple targets. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or another target of interest. Exemplary binding moieties of this disclosure include an antibody, a Fab^, F(ab^)2, Fab, Fv, rIgG, scFv, hcAb (heavy chain antibody), a single domain antibody, VHH, VNAR, sdAb, nanobody, receptor ectodomain or ligand-binding portions thereof, or ligand (e.g., cytokines, chemokines). An “Fab” (antigen binding fragment) is the part of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain via an inter-chain disulfide bond. In other embodiments, a binding moiety comprises a ligand-binding domain of a receptor or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. A variety of assays are known for identifying binding moieties of this disclosure that specifically bind a particular target, including Western blot, ELISA, biolayer interferometry, and surface plasmon resonance. A binding moiety, such as a binding moiety comprising immunoglobulin light and heavy chain variable domains (e.g., scFv), can be incorporated into a variety of protein scaffolds or structures as described herein, such as an antibody or an antigen binding fragment thereof, a scFv-Fc fusion protein, or a fusion protein comprising two or more of such immunoglobulin binding domains. [00280] The fundamental ability of the tLNP to deliver a payload into the cytoplasm of a cell is agnostic with respect to, and does not depend upon, a particular binding specificity. Of course, a binding moiety is a determinant of which cells a payload is delivered into. There are many known antibodies with specificity for one or another cell surface marker associated with particular cell type(s) that could be used as the target of the binding moiety on a disclosed tLNP and there are several sources that have compiled such information. An excellent source of information about antibodies for which an International Non-proprietary Name (INN) has been proposed or recommended is Wilkinson & Hale, MAbs 14(1):2123299, 2022, including its Supplementary Tables, which is incorporated by reference herein for all that it teaches about individual antibodies and the various antibody formats that can be constructed. US11,326,182 and especially its Table 9 Cancer, Inflammation and Immune System Antibodies, is a source of sequence and other information for a wide range of antibodies including many that do not have an INN and is incorporated herein by reference for all that it teaches about individual antibodies. Sequence information is not always readily available for antibodies mentioned in the art, even when commercially available. This is not necessarily an impediment to their use. Where the antibody or a cell line is commercially available or obtainable from its originator it can be used as the binding moiety of tLNP without any need for sequence information. Even where sequence information is needed, it is well within the capabilities of the skilled artisan to sequence the antibody protein (or have it done by a contract laboratory) so that the antibody’s variable region can be incorporated into a scFv, a diabody, a minibody, or some other antibody format, or be humanized. In choosing among available antibodies in the art for the development of an agent to be used in humans, a human antibody is preferred to a humanized antibody is preferred to a non- human antibody, other factors being equal. Other factors can include stability and ease of production of the antibody, affinity of the antibody, and cross-reactivity for the cognate antigen in model species to be used in product development. [00281] In some embodiments, a binding moiety can be an antibody or an antigen-binding portion thereof; an antigen; a ligand-binding domain of a receptor; or a receptor ligand. In some embodiments, a binding moiety can have more than one specificity including, for example, bispecific or multispecific binders. [00282] In some embodiments, a binding moiety comprises an antibody or an antigen- binding portion thereof. As used herein, “antibody” refers to a protein comprising an immunoglobulin domain having hypervariable regions determining the specificity with which the antibody binds antigen, termed complementarity determining regions (CDRs). The term antibody can thus refer to intact or whole antibodies as well as antibody fragments and constructs comprising an antigen binding portion of a whole antibody. While the canonical natural antibody has a pair of heavy and light chains, camelids (from camels, alpacas, llamas, etc.) produce antibodies with both the canonical structure and antibodies comprising only heavy chains. The variable region of the camelid heavy chain-only antibody has a distinct structure with a lengthened CDR3 referred to as VHH or, when produced as a fragment, a nanobody. Antigen binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fv, single-chain Fv (scFv), diabodies, and VH. Such elements can be combined to produce bi- and multi-specific reagents, including various immune cell engagers, such as BiTEs (bi-specific T-cell engagers). The term “monoclonal antibody” arose out of hybridoma technology but is now used to refer to any singular molecular species of antibody regardless of how it was originated or produced. Antibodies can be obtained through immunization, selection from a naïve or immunized library (for example, by phage display), alteration of an isolated antibody-encoding sequence, or any combination thereof. Numerous antibodies that can be used as binding moieties are known in the art. An excellent source of information about antibodies for an International Non-proprietary Name (INN) has been proposed or recommended, including sequence information, is Wilkinson & Hale, 2022, MAbs 14(1):2123299, including its Supplementary Tables, which is incorporated by reference herein for all that it teaches about individual antibodies and the various antibody formats that can be constructed. U.S. Patent No.11,326,182 and especially its Table 9 entitled “Cancer, Inflammation and Immune System Antibodies,” is a source of sequence and other information for a wide range of antibodies including many that do not have an INN and is incorporated herein by reference for all that it teaches about individual antibodies. WO2024040195A1 is also a source of sequence and other information for a wide range of antibodies with specificity for various cell surface antigens of immune system cells and cancer cells and is incorporated herein by reference for all that it teaches about individual antibodies and the antigens they bind. [00283] An antibody or other binding moiety (or a fusion protein thereof) “specifically binds” a target if it binds the target with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/Molar or 1/M) equal to or greater than 105 M-1, while not significantly binding other components present in a test sample. Binding domains (or fusion proteins thereof) can be classified as “high affinity” binding domains (or fusion proteins thereof) and “low affinity” binding domains (or fusion proteins thereof). “High affinity” binding domains refer to those binding domains with a Ka of at least 108 M-1, at least 109 M-1, at least 1010 M-1, at least 1011 M-1, at least 1012 M-1, or at least 1013 M-1, preferably at least 108 M-1 or at least 109 M-1. “Low affinity” binding domains refer to those binding domains with a Ka of up to 108 M-1, up to 107 M-1, up to 106 M-1, up to 105 M-1. Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10-5 M to 10-13 M). Affinities of binding domain polypeptides and fusion proteins according to this disclosure can be readily determined using conventional techniques (see, e.g., Scatchard et al., 1949, Ann. N.Y. Acad. Sci.51:660; and U.S. Patent Nos.5,283,173, 5,468,614, or the equivalent). [00284] A diabody is a type of scFv dimer in which each chain consists of the VH and VL regions connected by a small peptide linker that is too short to allow pairing between the two domains of the same chain. This arrangement forces the VH of one chain to pair with the VL of a second chain thereby forming a bivalent, and often bispecific, dimer. A BiTE is a fusion protein having two scFvs of different antibodies, usually an antibody for a tumor-associated antigen and antibody for CD3, on a single peptide chain, thus forming a cytolytic synapse between T cells and target antigen-bearing cells. The term "antigen-binding portion" can refer to a portion of an antibody as described that possesses the ability to specifically recognize, associate, unite, or combine with a target molecule. An antigen-binding portion includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a specific antigen. Thus, antibodies and antigen-binding portions thereof constitute means for binding to the surface molecule on a cell. In various embodiments, the cell can be an immune cell, a leukocyte, a lymphocyte, a monocyte, a stem cell, an HSC or an MSC, according to the specificity of the antibody. [00285] In some embodiments, the antibody or antigen-binding portion thereof can be derived from a mammalian species, for example, mice, rats, or human. Antibody variable regions can be those arising from one species, or they can be chimeric, containing segments of multiple species possibly further altered to optimize characteristics such as binding affinity or low immunogenicity. For human applications, it is desirable that the antibody has a human sequence. In the cases where the antibody or antigen-binding portion thereof is derived from a non-human species, the antibody or antigen-binding portion thereof can be humanized to reduce immunogenicity in a human subject. For example, if a human antibody of the desired specificity is not available, but such an antibody from a non-human species is, the non- human antibody can be humanized, e.g., through CDR grafting, in which the CDRs from the non-human antibody are placed into the respective positions in a framework of a compatible human antibody. Less preferred is an antibody in which only the constant region of the non- human antibody is replaced with human sequence. Such antibodies are commonly referred to as chimeric antibodies in distinction to humanized antibodies. [00286] In some embodiments, the antibody or antigen-binding portion thereof is non- immunogenic. In some embodiments, the antibody can be modified in its Fc region to reduce or eliminate secondary functions, such as FcR engagement, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement- dependent cytotoxicity (CDC); this is often referred to as an Fc silenced antibody. [00287] A binding moiety density on the LNP (or tLNP) can be defined according to the ratio of antibody (binder) to mRNA (w/w) either based on the amount of antibody input in the conjugation reaction or as measured in the LNP or (tLNP) after conjugation. For an intact antibody (e.g., whole IgG), in some embodiments, preferred ratios are about 0.3 to about 1.0, about 0.3 to about 0.7, about 0.3 to about 0.5, about 0.5 to about 1.0, and about 0.5 to about 0.7 for either the input or final measured binder ratio. In certain embodiments, a LNP (or tLNP) has an antibody ratio of 0.3 to 1.0, 0.3 to 0.7, 0.3 to 0.5, 0.5 to 1.0, and 0.5 to 0.7 for either the input or final measured binder ratio. In some embodiments, if the binder is different in size from an intact antibody (for example a scFv, diabody, or minibody, etc.) the w/w ratio is adjusted for the different size of the binder. [00288] In certain embodiments, a LNP or tlNP comprise a binding moiety that is a F(ab’) or F(ab’) analog. F(ab’) and F(ab’)-like formats offer certain advantages as targeting moieties for a tLNP. Although any antibody fragment with a structure similar to or derived from that of a classical, proteolytically produced F(ab’) is often referred to as an F(ab’), the term “F(ab’) analog” has been adopted herein to refer to engineered sequences comprising amino acid substitutions and/or that have been truncated and to distinguish them from the paradigmatic natural sequence. F(ab’) are smaller than whole antibodies which can be advantageous in manufacturing. When used as a targeting moiety on a tLNP their antigen binding domain is further from the LNP surface than, for example, a scFv, which can facilitate interaction with the target cell surface. F(ab’) molecules have cysteine residues in the partial hinge region that can be readily conjugated to a functionalized PEG-lipid (for example, a maleimide- functionalized PEG-lipid). Moreover, the F(ab’) can be engineered so that there is unique accessible cysteine enabling for site-specific conjugation which is desirable for product consistency. This can be accomplished with recombinant DNA technology by truncating the hinge region of the F(ab’) or by changing cysteine residues to another amino acid, such as serine, or both. [00289] The hinge region cysteines can form a cystine with another F(ab’) molecule forming an F(ab’)2 which would make the cysteine unavailable for conjugation to an LNP (more specifically, a functionalized lipid thereof). This can be prevented by processing the F(ab’) under mildly reducing conditions, however, this poses a risk of disrupting the interchain disulfide bond between CL and CH1. That risk can be obviated by relocating the interchain bond to a less accessible region in the molecule. [00290] Some aspects combine constant regions of an F(ab’) or F(ab’) analog with a humanized immunoglobulin antigen binding domain derived from the anti-CD8α antibody CT8 as disclosed herein. Humanized anti-CD8 antigen binding domains and antibodies and F(ab’) analog design useful as LNP targeting moieties are disclosed, for example, in International Patent Application No. PCT/US2024/060426, filed December 16, 2024, which is incorporated for reference for all that it teaches about anti-CD8 antigen binding domains and antibodies and F(ab’) analog designs. [00291] Some aspects combine constant regions of an F(ab’) analog with the antigen binding domain of an anti-CD8 antibody. In some embodiments, the anti-CD8 antigen binding domain recognizes the CT8 epitope. In some embodiments, the anti-CD8 antigen binding domain is derived from YTC182.20, TRX2, or CT8. In some embodiments, the anti-CD8 antigen binding domain comprises a humanized immunoglobulin antigen binding domain derived from the anti-CD8α antibody CT8 as disclosed herein. [00292] In some aspects, an F(ab’) analog engineered as disclosed herein is conjugated to an LNP but is generic with respect to the variable domains of the F(ab’) analog and its specificity. [00293] In some aspects, an F(ab’) or F(ab’) analog constant regions are combined with the antigen binding domain of an anti-CD8 antibody which is conjugated to an LNP. In some embodiments, the anti-CD8 antigen binding domain recognizes the CT8 epitope. In some embodiments, the anti-CD8 antigen binding domain is derived from YTC182.20, TRX2, or CT8. In some embodiments, the anti-CD8 antigen binding domain comprises a humanized immunoglobulin antigen binding domain derived from the anti-CD8α antibody CT8 as disclosed herein. [00294] With respect to these forgoing aspects, in some embodiments, the F(ab’) analog, as appropriate, comprises a relocated interchain disulfide bond, for example, a Cκ S162C substitution paired with an IgG1 or IgG4 CH1 F174C substitution. In further embodiments, one, the other, or both cysteines involved in forming the native interchain disulfide bond are mutated, for example, Cκ C214S, IgG1 C233S, or IgG4 CH1 C127S. [00295] In certain embodiments, a LNP or tLNP comprises a binding moiety derived from an anti-CD40* antibody, an anti-LRRC15† antibody, an anti-CTSK antibody, an anti- ADAM12 antibody, an anti-ITGA11 antibody, an anti-FAP*† antibody, an anti-NOX4 antibody, an anti-SGCD antibody, an anti-SYNDIG1 antibody, an anti-CDH11 antibody, an anti-PLPP4 antibody, an anti-SLC24A2 antibody, an anti-PDGFRB* antibody, an anti-THY1 antibody, an anti-ANTXR1 antibody, an anti-GAS1 antibody, an anti-CALHM5 antibody, an anti-SDC1* antibody, an anti-HER2*† antibody, an anti-TROP2*† antibody, an anti-MSLN* antibody, an anti-Nectin4† antibody, or an anti-MUC16*† antibody. In further embodiments, a LNP (or tLNP) comprises a binding moiety specific for an immune cell antigen selected from CD1, CD2*†, CD3*†, CD4*†, CD5†, CD7†, CD8†, CD11b, CD14†, CD16, CD25†, CD26*, CD119, CD126, CD137 (41BB)†, CD150, CD153, CD161, CD166, CD183 (CXCR3), CD183 (CXCR5), CD223 (LAG-3)*†, CD254, CD275, CD45RA, CTLA-4*†*†, DEC205, OX40†, PD-1*†, GITR†, TIM-3*†, , FasL*, IL18R1, ICOS (CD278), leu-12, TCR†, TLR1, TLR2†, TLR3*, TLR4†, TLR6, TREM2, NKG2D, CCR, CCR1 (CD191), CCR2 (CD192)*†, CCR4(CD194)*†, CCR6(CD196), CCR7, low affinity IL-2 receptor†, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, and IL-21 receptor. In further embodiments, a tLNP comprises a binding moiety specific for an HSC surface molecule selected from CD117†, CD34*, CD44*, CD90 (Thy1) , CD105, CD133, BMPR2, and Sca-1; or specific for an MSC surface molecules selected from CD70*, CD105, CD73, Stro-1, SSEA-3, SSEA-4, CD271, CD146, GD2*†, SUSD2, Stro-4, MSCA-1, CD56, CD200*†, PODXL, CD13, CD29*, CD44*, and CD10. In various embodiments, a binding moiety is an antibody or antigen-binding portion thereof. (* indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in US Patent 11,326,182B2 Table 9 or 10. †indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in Wilkinson & Hale, 2022. Both references cited and incorporated by reference above. indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in the Therapeutic Antibody Database (TABS) at tabs.craic.com). Other suitable antibodies can be found in Appendix A or in International Patent Publication No. WO2024040195A1, filed August 17, 2023, each of which is incorporated herein by reference for all that it teaches about individual antibodies and the antigens they bind. [00296] The following paragraphs provide non-exhaustive examples of known antibodies that bind to cell surface markers/antigens on immune cells (lymphocytes and monocytes) and stem cells (HSC and MSC). These antibodies or the antigen binding domains thereof can be used as binding moieties to target the disclosed LNP. Similarly, these antibodies can contribute their antigen binding domains to immune cell reprogramming agents such as CARs and ICEs. While typically an immune cell reprogramming agent is expressed in an immune cell, one call also express a biological response modifier (conditioning agent) or an immune cell reprogramming agent, such as an ICE, in a tumor cell. The immune and stem cell surface markers that can serve as a targeted antigen of a tLNP can also usefully be a target of an immune cell reprogramming agent when the cell expressing that antigen has a role in the pathology of some disease or condition. Collectively these antibodies and polypeptides comprising the antigen binding domains thereof constitute means for binding cell surface markers or means for binding immune and stem cells. [00297] In some embodiments, CD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD2 antibody. CD2 contains three well-characterized epitopes (T11.1, T11.2, and T11.3/CD2R). T11.3/CD2R are membrane proximal and exposure is increased upon T cell activation and CD2 clustering. Accordingly, in some such embodiments, the anti-CD2 antigen binding domain is derived from, RPA-2.10; OKT11, UMCD2, 0.1, and 3T4-8B5 (T11.1 epitope); 9.6 and 1OLD2-4C1 (T11.2 epitope); 1Mono2A6 (T11.3 epitope), siplizumab (T11.2/T11.3 epitope), HuMCD2, TS2/18, TS1/8, AB75, LT-2, T6.3, MEM-65, OTI4E4, or an antigen-binding portion thereof. Additionally, the ligand of CD2, CD58 (LFA-3) can be used as a CD2 binding moiety as can alefacept, a CD58-Fc fusion. Each of these constitutes a means for binding CD2 (Li et al., 1996, J Mol Biol.263:209-26; Binder et al., 2020, Front Immunol.9:11:1090). [00298] In some embodiments, CD3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from muromonab-CD3 (OKT3), teplizumab, otelixizumab, visilizumab, cevostamab, teclistamab, elranatamab pavurutamab, vibecotamab, odronextamab, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD3. [00299] In some embodiments, CD4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ibalizumab, inezetamab, semzuvolimab, zanolimumab, tregalizumab, UB-421, priliximab, MTRX1011A, cedelizumab, clenoliximab, keliximab, M-T413, TRX1, hB-F5, MAX.16H5, IT208, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD4. [00300] In some embodiments, CD5 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD5 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from 5D7, UCHT2, L17F12, H65, HE3, OKT1, MAT304, as well as those disclosed in WO1989006968, WO2008121160, US8,679,500, WO2010022737, WO2019108863, WO2022040608, or WO2022127844, each of which is incorporated by reference for all that they teach about anti-CD5 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD5. [00301] In some embodiments, CD7 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD7 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from TH-69, 3A1E, 3A1F, Huly- m2, WT1, YTH3.2.6, T3-3A1, grisnilimab, as well as those disclosed in US10,106,609, WO2017213979, WO2018098306, US11447548, WO2022136888, WO2020212710, WO2021160267, WO2022095802, WO2022095803, WO2022151851, or WO2022257835 each of which is incorporated by reference for all that they teach about anti-CD7 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD7. [00302] In some embodiments, CD8 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD8 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from crefmirlimab (IAB22M), 3B5, SP-16, LT8, 17D8, MEM-31, MEM-87, RIV11, UCHT4, YTC182.20, RPA-T8, OKT8, SK1, 51.1, TRX2, MT807-R1, HIT8α, C8/144B, RAVB3, SIDI8BEE, BU88, EPR26538-16, 2ST8.5H7, as well as those disclosed in US10,414,820, WO2015184203, WO2017134306, WO2019032661, WO2020060924, US10,730,944, WO2019033043, WO2021046159, WO2021127088, WO2022081516, US11,535,869, or WO2023004304 each of which is incorporated by reference for all that they teach about anti-CD8 antibodies and their properties, or an antigen-binding portion thereof. Additionally, humanized anti-CD8 antibodies are described in U.S. Provisional Patent Application Number 63/610,917, filed on December 15, 2023, U.S. Provisional Patent Application Number 63/654,930, filed on May 31, 2024, U.S. Provisional Patent Application Number 63/708,461, filed on October 17, 2024, and International Patent Application Number PCT/US2024/060426, filed on December 16, 2024, each of which is incorporated by reference for all that it teaches about these humanized anti-CD8 antibodies and their properties, or an antigen-binding portion thereof. Each of the foregoing anti-CD8 antibodies constitutes a means for binding CD8. [00303] In some embodiments, a tLNP is targeted to CD10 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD10 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from the one produced by the hybridoma represented by Accession No. NITE BP-02489 (disclosed in WO2018235247 which is incorporated by reference for all that they teach about anti-CD10 antibodies and their properties), FR4D11, or REA877, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD10. [00304] In some embodiments, CD11b is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD11b antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ASD141 or MAB107 as well as those disclosed in US20150337039, US10,738,121, WO2016197974, US10,919,967, or WO2022147338 each of which is incorporated by reference for all that they teach about anti-CD11b antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD11b. [00305] In some embodiments, CD13 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD13 antibody. CD13 is also known as aminopeptidase N (APN). Accordingly, in some such embodiments, the antigen binding domain is derived from MT95-4 or Nbl57 (disclosed in WO2021072312 which is incorporated by reference for all that they teach about anti-CD13 antibodies and their properties), as well as those disclosed in WO2023037015 which is incorporated by reference for all that it teaches about anti-CD13 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD13. [00306] In some embodiments, CD14 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD14 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from atibuclimab or r18D11 as well as those disclosed in WO2018191786 or WO2015140591 each of which is incorporated by reference for all that they teach about anti-CD14 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD14. [00307] In some embodiments, CD16a is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD16a antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from AFM13, sdA1, sdA2, or hu3G8-5.1-N297Q as well as those disclosed in US11535672, WO2018158349, WO2007009065, US10385137, WO2017064221, US10,758,625, WO2018039626, WO2018152516, WO2021076564, WO2022161314, or WO2023274183 each of which is incorporated by reference for all that they teach about anti-CD16A antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD16a. [00308] In some embodiments, CD25 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD25 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from daclizumab, basiliximab, camidanlumab, tesirine, inolimomab, RO7296682, HuMax-TAC, CYT-91000, STI-003, RTX-003, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD25. [00309] In some embodiments, CD28 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD28 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from GN1412, acazicolcept, lulizumab, prezalumab, theralizumab, FR104CD, and davoceticept, as well as those disclosed in US8,454,959, US8,785,604, US11,548,947, US11,530,268, US11,453,721, US11,591,401, WO2002030459, WO2002047721, US20170335016, US20200181260, US11608376, WO2020127618, WO2021155071, or WO2022056199 each of which is incorporated by reference for all that they teach about anti-CD28 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD28. [00310] In some embodiments, CD29 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD29 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from OS2966, 6D276, 12G10, REA1060, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD29. [00311] In some embodiments, CD32A is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD32A antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from VIB9600, humanized IV.3, humanized AT-10, or MDE-8 as well as those disclosed in US9,688,755, US9,284,375, US9,382,321, US11306145, or WO2022067394 each of which is incorporated by reference for all that they teach about anti-CD32A antibodies and their properties, or an antigen- binding portion thereof. Each of these constitutes a means for binding CD32A. [00312] In some embodiments, CD34 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD34 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from h4C8, 9C5, 2E10, 5B12, REA1164, C5B12, C2e10, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD34. [00313] In some embodiments, CD40 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD40 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from cifurtilimab, sotigalimab, iscalimab, dacetuzumab, selicrelumab, bleselumab, lucatumumab, or mitazalimab as well as those disclosed in US10633444, each of which is incorporated by reference for all that they teach about anti-CD40 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD40. [00314] In some embodiments, CD44 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD44 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from RO5429083, VB6-008, PF-03475952, or RG7356, as well as those disclosed in WO2008144890, US8,383,117, WO2008079246, US20100040540, WO2015076425, US9,220,772, US20140308301, WO2020159754, WO2021160269, WO2021178896, WO2022022749, WO2022022720, or WO2022243838, each of which is incorporated by reference for all that they teach about anti-CD44 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD44. [00315] In some embodiments, CD45 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD45 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from apamistamab, BC8- B10, as well as those disclosed in WO2023183927, WO2023235772, US7,825,222, WO2017009473, WO2021186056, US9,701,756, US9,701,756, WO2020092654, WO2022040088, WO2022040577, WO2022064191, WO2022063853, or WO2024064771, each of which is incorporated by reference for all that they teach about anti-CD45 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD45. [00316] In some embodiments, CD56 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD56 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from lorvotuzumab, adcitmer, or promiximab, as well as those disclosed in WO2012138537, US10,548,987, US10,730,941, or US20230144142, each of which is incorporated by reference for all that they teach about anti-CD56 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD56. [00317] In some embodiments, CD64 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD64 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from HuMAb 611 or H22 as well as those disclosed in US7,378,504, WO2014083379, US20170166638, or WO2022155608 each of which is incorporated by reference for all that they teach about anti- CD64 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD64. [00318] In some embodiments, CD68 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD68 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from Ki-M7, PG-M1, 514H12, ABM53F5, 3F7C6, 3F7D3, Y1/82A, EPR20545, CDLA68-1, LAMP4-824, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD68. [00319] In some embodiments, CD70 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD70 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from cusatuzumab, vorsetuzumab, MDX-1203, MDX-1411, AMG-172, SGN-CD70A, ARX305, PRO1160, as well as those disclosed in US9,765,148, US8,124,738, IS10,266,604, WO2021138264, US9,701,752, US10,108,123, WO2014158821, US10,689,456, WO2017062271, US11,046,775, US11,377,500, WO2021055437, WO2021245603, WO2022002019, WO2022078344, WO2022105914, WO2022143951, WO2023278520, WO2022226317, WO2022262101, US11,613,584, or WO2023072307, each of which is incorporated by reference for all that they teach about anti-CD70 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD70. [00320] In some embodiments, CD73 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD73 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from oleclumab, uliledlimab, mupadolimab, AK119, IBI325, BMS-986179, NZV930, JAB-BX102, Sym024, TB19, TB38, HBM1007, 3F7, mAb19, Hu001-MMAE, IPH5301, or INCA00186, as well as those disclosed in US9,938,356, US10,584,169, WO2022083723, WO2022037531, WO2021213466, WO2022083049, US10,822,426, WO2021259199, US10,100,129, US11,312,783, US11,174,319, US11,634,500, WO2021138467, WO2017118613, US9,388,249, WO2020216697, US11180554, US11,530,273, WO2019173692, WO2019170131, US11,312,785, WO2020098599, WO2020143836, WO2020143710, US11,034,771, US11,299,550, WO2020253568, WO2021017892, WO2021032173, WO2021032173, WO2021097223, WO2021205383, WO2021227307, WO2021241729, WO2022096020, WO2022105881, WO2022179039, WO2022214677, or WO2022242758, each of which is incorporated by reference for all that they teach about anti-CD73 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD73. [00321] In some embodiments, CD90 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD90 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REA897, OX7, 5E10, K117, L127, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD90. [00322] In some embodiments, CD105 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD105 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from carotuximab, TRC205, or huRH105, as well as those disclosed in US8,221,753, US9,926,375, WO2010039873, WO2010032059, WO2012149412, WO2015118031, WO2021118955, US20220233591, or US20230075244, each of which is incorporated by reference for all that they teach about anti-CD105 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD105. [00323] In some embodiments, CD117 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD117 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from briquilimab, barzolvolimab, CDX-0158, LOP628, MGTA-117, NN2101, CK6, JSP191, Ab85, 104D2, or SR1, as well as those disclosed in US7,915,391, WO2022159737, US9540443, WO2015050959, US9,789,203, US8,552,157, US10,406,179, US9,932,410, WO2019084067, WO2020219770, US10,611,838, WO2020076105, WO2021107566, US11,208,482, WO2021044008, WO2021099418, WO2022187050, or WO2023026791, WO2021188590, each of which is incorporated by reference for all that they teach about anti-CD117 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD117. [00324] In some embodiments, CD133 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD133 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from AC133, 293C3, CMab- 43, or RW03, as well as those disclosed in WO2018045880, US8,722,858, US9,249,225, WO2014128185, US10,711,068, US10,106,623, WO2018072025, or WO2022022718, each of which is incorporated by reference for all that they teach about anti-CD133 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD133. [00325] In some embodiments, CD137 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD137 antibody. CD137 is also known as 4-1BB. Accordingly, in some such embodiments, the antigen binding domain is derived from YH004, urelumab (BMS-663513), utomilumab (PF-05082566), ADG106, LVGN6051, PRS-343, as well as those disclosed in WO2005035584, WO2012032433, WO2017123650, US11,203,643, US11,242,395, US11,555,077, US20230067770, US11,535,678, US11,440,966, WO2019092451, US10,174,122, US11,242,385, US10,716,851, WO2020011966, WO2020011964, or US11,447,558, each of which is incorporated by reference for all that they teach about CD137 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD137. [00326] In some embodiments, CD146 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD146 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from imaprelimab, ABX- MA1, huAA98, M2H, or IM1-24-3, as well as those disclosed in US10,407,506, US10,414,825, US6,924,360, US9,447,190, WO2014000338, US9,782,500, WO2018220467, US11,427,648, WO2019133639, WO2019137309, WO2020132190, or WO2022082073, each of which is incorporated by reference for all that they teach about CD146 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD146. [00327] In some embodiments, CD166 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD166 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from praluzatamab, AZN- L50, REA442, or AT002, as well as those disclosed in US10,745,481, US11,220,544, or WO2008117049, each of which is incorporated by reference for all that they teach about CD166 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD166. [00328] In some embodiments, CD200 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD200 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from samalizumab, OX-104, REA1067, B7V3V2, HPAB-0260-YJ, or TTI-CD200, as well as those disclosed in WO2007084321 or WO2019126536, each of which is incorporated by reference for all that they teach about CD200 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD200. [00329] In some embodiments, CD205 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD205 antibody. CD205 is also known as DEC205. Accordingly, in some such embodiments, the antibody comprises 3G9- 2D2 (a component of CDX-1401) or LY75_A1 (a component of MEN1309) as well as those disclosed in US8,236,318, US10,081,682, or US11,365,258, each of which is incorporated by reference for all that they teach about anti-CD205 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD205. [00330] In some embodiments, CD271 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CD271 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REA844 or REAL709 as well as those disclosed in WO2022166802 which is incorporated by reference for all that it teaches about anti-CD271 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding CD271. [00331] In some embodiments, BMPR2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-BMPR2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from TAB-071CL (Creative Biolabs catalog no.) as well as those disclosed in US11,292,846 or WO2021174198, each of which is incorporated by reference for all that they teach about anti-BMPR2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding BMPR2. [00332] In some embodiments, claudin 18.2 (CLDN 18.2) is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-claudin 18.2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from zolbetuximab, osemitamab, RC118, IBI-343, AZD0901, M108, SYSA1801, TORL-2- 307-ADC, LM-302, ASKB589, gresonitamab, SPX-101, SKB315, Q-1802, GIVASTOMIG, LCAR-C18S, SOT102, CT041 as well as those disclosed in WO2013167259, WO2021032157, WO2021254481, WO2022007808, WO2021008463, WO2022111616, WO2018006882, WO2020147321, WO2019219089, US20200040101, WO2020025792, WO2020139956, WO2020135201, US20240228610, WO2021218874, WO2021027850, WO2021129765, WO2022068854, WO2021111003, each of which is incorporated by reference for all that it teaches about anti- claudin 18.2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding claudin 18.2. [00333] In some embodiments, CTLA-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-CTLA-4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from botensilimab, ipilimumab, nurulimab, quavonlimab, tremelimumab, zalifrelimab, ADG116, ADG126, ADU- 1604, AGEN1181, BCD-145, BMS-986218, BMS-986249, BT-007, CS1002, GIGA-564, HBM4003, IBI310 JK08, JMW-3B3, JS007, KD6001, KN044, ONC-392, REGN4659, TG6050, XTX101, YH001, or an antigen-binding portion thereof. Each of these constitutes a means for binding CTLA-4. [00334] In some embodiments, GD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GD2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from dinutuximab, ganglidiximab, naxitamab, nivatrotamab, EMD 273063, hu14.18k322A, MORAb-028, 3F8BiAb, BCD-245, KM666, ATL301, Ektomab, as well as those disclosed in US9,777,068, US9,315,585, WO2004055056, US9,617,349, US9,493,740, US20210002384, US8507657, WO2001023573, WO2012071216, WO2018010846, US8,951,524, WO2023280880, US9,856,324, WO2015132604, WO2017055385, WO2019059771, WO2020020194, or an antigen-binding portion thereof. Each of these constitutes a means for binding GD2. [00335] In some embodiments, GITR is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GITR antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ragifilimab, TRX518, MK- 4166, AMG 228, MEDI1873, BMS-986156, REGN6569, ASP1951, MK-1248, FRA154, GWN323, JNJ-64164711, ATOR-1144, or an antigen-binding portion thereof. Each of these constitutes a means for binding GITR. [00336] In some embodiments, a low affinity IL-2 receptor is a targeted cell surface antigen (CD122 and/or CD132) and a binding moiety comprises the antigen binding domain of an anti-IL-2 receptor antibody. Accordingly, in some such embodiments, the antiCD122 antibody comprises ANV419, FB102, MiK-Beta-1 and the anti CD122 antibodies disclosed in WO2011127324, WO2017021540, WO2022212848, WO2022221409, WO2023078113, US20230272090, WO2024073723, or an antigen-binding portion thereof. Accordingly, in some such embodiments, the anti-CD132 antibody comprises REGN7257 and the anti- CD132 antibodies disclosed in WO2020160242, WO2017021540, WO2022212848, WO2023078113, US20230272089, or an antigen-binding portion thereof. Each of these constitutes a means for binding the low affinity IL-2 receptor (CD122 or CD132, as appropriate), [00337] In some embodiments, a high affinity IL-2 receptor is a targeted cell surface antigen (CD25) and a binding moiety comprises the antigen binding domain of an anti-IL-2 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from daclizumab, basiliximab, camidanlumab, vopitug, inolimomab, HuMAx-TAC, Xenopax, STI-003, RA8, RTX-003, and the anti-CD25 antibodies disclosed in WO2023031403, WO2006108670, WO2019175223, WO2019175215, WO2019175226, WO2004045512, WO2022104009, WO2020102591, or an antigen-binding portion thereof. Each of these constitutes a means for binding the high affinity IL-2 receptor (CD25). [00338] In some embodiments, IL-7 receptor (CD127) is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-7 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from PF-06342674, GSK2618960, OSE-127, lusvertikimab, bempikibart, and the anti-CD127 antibodies disclosed in WO2011104687, WO2011094259, WO2013056984, WO2015189302, WO2017062748, WO2020154293, WO2020254827, WO2021222227, WO2023201316, or an antigen-binding portion thereof. Each of these constitutes a means for binding the CD127. [00339] In some embodiments, IL-12 receptor is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-12 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from CBYY- I0413, REA333, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-12 receptor. [00340] In some embodiments, IL-15 receptor α is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-15 receptor α antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MAB1472-100, MAB5511, JM7A4, 5E3E1, JM7A4, 2639B, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-15 receptor α. [00341] In some embodiments, IL-18 receptor α is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-18 receptor α antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from H44, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-18 receptor α. [00342] In some embodiments, IL-21 receptor is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-IL-21 receptor antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from 1D1C2, 19F5, 18A5, REA233, or an antigen-binding portion thereof. Each of these constitutes a means for binding the IL-21 receptor α. [00343] In some embodiments, LAG-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-LAG-3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from relatlimab, tebotelimab, favezelimab, fianlimab, miptenalimab, HLX26, ieramilimab, GSK2831781, INCAGN2385, RO7247669, encelimab, FS118, SHR-1802, Sym022, IBI110, IBI323, bavunalimab, EMB-02, ABL501, INCA32459, AK129, or an antigen-binding portion thereof. Each of these constitutes a means for binding LAG-3. [00344] In some embodiments, MSCA-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti- MSCA-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REAL219, W8B2, X9C3, or an antigen-binding portion thereof. Each of these constitutes a means for binding MSCA-1. [00345] In some embodiments, OX40 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-OX40 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MEDI6469, ivuxolimab, rocatinlimab, GSK3174998, BMS-986178, vonlerizumab, INCAGN1949, tavolimab, BGB-A445, INBRX-106, BAT6026, telazorlimab, ATOR-1015, MEDI6383, cudarolimab, FS120, HFB301001, EMB-09, HLX51, Hu222, ABM193, or an antigen-binding portion thereof. Each of these constitutes a means for binding OX40. [00346] In some embodiments, PD-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-PD-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from nivolumab, pembrolizumab, camrelizumab, torpalimab, sintilimab, tislelizumab, cemiplimab, spartalizumab, serplulimab, cadonilimab, penpulimab, dostarlimab, zimberelimab, retifanlimab, pucotenlimab, pidilizumab, pidilizumab, balstilimab, ezabenlimab, AK112, geptanolimab, cetrelimab, prolgolimab, tebotelimab, sasanlimab, SG001, vudalimab, MEDI5752, rulonilimab, peresolimab, IBI318, budigalimab, MEDI0680, pimivalimab, QL1706, AMG 404, RO7121661, lorigerlimab, nofazinlimab, sindelizumab, or an antigen-binding portion thereof. Each of these constitutes a means for binding PD-1. [00347] In some embodiments, PODXL is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-PODXL antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MAI1738, HPAB- 3334LY, HPAB-MO612-YC, REA246, REA157, or an antigen-binding portion thereof. Each of these constitutes a means for binding PODXL. [00348] In some embodiments, Sca-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Sca-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from CPP324-1-18, 2D4- C9-F1, AMM22070N, or an antigen-binding portion thereof. Each of these constitutes a means for binding SCA-1. [00349] In some embodiments, SSEA-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SSEA-3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from MC631, 2A9, 8A7, ND- 742, 3H420, as well as those disclosed in US11,643,456 or WO2021138378, each of which is incorporated by reference for all that they teach about anti-SSEA-3 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding SSEA-3. [00350] In some embodiments, SSEA-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SSEA-4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from ch28/11, REA101, MC-813-70, ND-942-80, as well as those disclosed in US11,446,379, US10,273,295, US11,643,456, WO2019190952, or WO2021044039, each of which is incorporated by reference for all that they teach about anti-SSEA-4 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding SSEA-4. [00351] In some embodiments, Stro-1 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Stro-1 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from STRO-1, TUSP-2, as well as those disclosed in US20130122022, which is incorporated by reference for all that it teaches about anti-Stro-1 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding Stro-1. [00352] In some embodiments, Stro-4 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-Stro-4 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from STRO-4, efungumab, 4C5, as well as those disclosed in US7,722,869, US20110280881, US9,115,192, US10,273,294, US10,457,726, WO2023091148, each of which is incorporated by reference for all that they teach about anti-Stro-4 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding Stro-4 (also known as heat shock protein-90). [00353] In some embodiments, SUSD2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-SUSD2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from REA795, CBXS-3571, CBXS-1650, CBXS-1989, CBXS-1671, CBXS1990, CBXS-3676, 1279B, EPR8913(2), W5C5, or an antigen-binding portion thereof. Each of these constitutes a means for binding SUSD2. [00354] In some embodiments, TIM-3 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-TIM-3 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from TQB2618, sabatolimab, cobolimab, RO7121661, INCAGN02390, AZD7789, surzebiclimab, LY3321367, Sym023, BMS-986258, SHR-1702, LY3415244, LB1410, or an antigen-binding portion thereof. Each of these constitutes a means for binding TIM-3. [00355] In some embodiments, TREM2 is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-TREM2 antibody. Accordingly, in some such embodiments, the antigen binding domain is derived from PI37012 as well as those disclosed in US10,508,148, US10,676,525, WO2017058866, US11,186,636, US11,124,567, WO2020055975, US11,492,402, WO2020121195, WO2023012802, WO2021101823, WO2023047100, WO2022032293, WO2022241082, WO2023039450, or WO2023039612, each of which is incorporated by reference for all that they teach about anti-TREM2 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding TREM2. [00356] In some embodiments, G protein-coupled receptor, class C, group 5, member D (GPRC5D) is a targeted cell surface antigen and a binding moiety comprises the antigen binding domain of an anti-GPRC5D antibody. Accordingly, in some such embodiments, the antigen binding domain of an anti-GPRC5D antibody is derived from talquetamab, forimtamig, BMS-986393, IBI-3003, QLS32015, SIM0500, or EPR28376-41, or is disclosed in WO2018017786, WO2016090329, WO2022174813, WO2023236889, WO2018147245, WO2024079015, WO2019154890, WO2021018859, WO2021018925, WO2020092854, WO2024031091, WO2020148677, WO2022175255, WO2022222910, WO2022247804, WO2022247756, WO2023078382, WO2023125728, WO2023143537, WO2024046239, or WO2024131962 each of which is incorporated by reference for all that they teach about anti- GPRC5D antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding GPRC5D. [00357] In some embodiments, FCRL5 (CD307E) is a targeted cell surface antigen and binding moiety comprises the antigen binding domain of an anti-FCRL5 antibody. Accordingly, in some such embodiments, the antigen binding domain of an anti-FCRL5 antibody is derived from cevostamab, 2A10H7, 307307, 2A10D6, EPR27365-87, EPR26948-19, or EPR26948-67, or is disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558 each of which is incorporated by reference for all that they teach about anti-FCRL5 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding FCRL5. [00358] In some embodiments, LRRC15 is a targeted cell surface antigen and binding moiety comprises the antigen binding domain of an anti-LRRC15 antibody. Accordingly, in some such embodiments, the antigen binding domain of an anti-LRRC15 antibody is derived from samrotamab or DUNP19 or is disclosed in WO2005037999, WO2021022304, WO2024081729, WO2021102332, WO2021202642, WO2022157094, or WO2024158047, each of which is incorporated by reference for all that they teach about anti-LRRC15 antibodies and their properties, or an antigen-binding portion thereof. Each of these constitutes a means for binding LRRC15. [00359] In still further embodiments, a tLNP is targeted to a tumor cell. In some embodiments, the tumor cell expresses one of the antigens described above and the tLNP is targeted to antigen expressing tumors using the same means as described above. In other embodiments the tLNP is targeted to some other tumor antigen, such as those enumerated in U.S. Provisional Application No.63/371,742, filed on August 17, 2022, entitled CONDITIONING FOR IN VIVO IMMUNE CELL ENGINEERING which is incorporated by reference for all that it teaches about the delivery of nucleic acids into tumor cells using tLNP that is not inconsistent with this disclosure. Nucleic Acid [00360] In certain embodiments, the disclosed LNP and tLNP comprise a payload comprising or consisting of one or more nucleic acid species. In some embodiments, the LNP or tLNP payload comprises only one nucleic acid species while in other embodiments the LNP or tLNP payload comprises multiple nucleic acid species, for example, 2, 3, or 4 nucleic acid species. For example, in embodiments in which the payload comprises a nucleic acid encoding a CAR or immune cell engager (ICE), the payload can comprise or consist of 1) a single nucleic acid species encoding a single species of CAR or ICE, 2) a single nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which each CAR and/or ICE has specificity for a same target antigen, 3) a single nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) such as a bicistronic or multicistronic mRNA in which at least one CAR and/or ICE has specificity for a different target antigen than the other(s), 4) two or more nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) in which each CAR and/or ICE has specificity for a same target antigen, 5) two or more nucleic acid species encoding 2 or more species of CAR or ICE (or a mixture of CAR and ICE) in which at least one CAR and/or ICE has specificity for a different target antigen than the other(s). When two or more CAR and/or ICE have specificity for a same target antigen, they can have specificity for different epitopes of the same target antigen. Further variations will be apparent to one of skill in the art (e.g., multiple bi- or multicistronic nucleic acids, nucleic acids encoding a TCR and the like). The nucleic acid can be RNA or DNA. The nucleic acid can be multicistronic, for example, bicistronic. [00361] In some embodiments, LNPs or tLNPs of this disclosure further comprise a nucleic acid payload. In various embodiments, a nucleic acid is an mRNA, a self-replicating RNA (also known as self-amplifying RNA), a circular RNA, a siRNA, a miRNA, DNA, a gene editing component (for example, a guide RNA, a tracr RNA, an sgRNA), a gene writing component, an mRNA encoding a gene or base editing protein, a zinc-finger nuclease, a TALEN, a CRISPR nuclease, such as Cas9, a DNA molecule to be inserted or serve as a template for repair), and the like, or a combination thereof. In some embodiments, the nucleic acid comprises small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO). In some embodiments, the nucleic acid comprises a self-replicating RNA or a circular RNA. In some embodiments, the mRNA encodes a reprogramming agent or comprises or encodes a conditioning agent. In some embodiments, the RNA (linear mRNA, circular, or self-replicating) comprises an miRNA binding site. In some embodiments, an mRNA encodes a chimeric antigen receptor (CAR). In other embodiments, an mRNA encodes a gene-editing or base-editing or gene writing protein. In some embodiments, a nucleic acid is a guide RNA. In some embodiments, an LNP or tLNP comprises both a gene- or base-editing or gene writing protein-encoding mRNA and one or more guide RNAs. CRISPR nucleases can have altered activity, for example, modifying the nuclease so that it is a nickase instead of making double-strand cuts or so that it binds the sequence specified by the guide RNA but has no enzymatic activity. Base-editing proteins are often fusion proteins comprising a deaminase domain and a sequence-specific DNA binding domain (such as an inactive CRISPR nuclease). [00362] In some embodiments, the reprogramming agent comprises an immune receptor (for example, a chimeric antigen receptor or a T cell receptor) or an immune cell engager (for example, a bispecific T cell engager (BiTE), a bispecific killer cell engager (BiKE), a trispecific kill cell engager (TriKE), a dual affinity retargeting antibody (DART), a TRIDENT (linking two DART units or a DART unit and a Fab domain), a macrophage engager (e.g., BiME), an innate cell engager, and the like). [00363] In some embodiments, the nucleic acid is an RNA, for example, mRNA, and the RNA comprises at least one modified nucleoside. In some embodiments, the modified nucleoside is pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methyluridine, N6- methyladenosine, 2’-O-methyluridine, or 2-thiouridine. In certain embodiments, all of the uridines are substituted with a modified nucleoside. Further disclosure of modified nucleosides and their use can be found in U.S. Patent No.8,278,036 which is incorporated herein by reference for those teachings. [00364] In some embodiments, the reprogramming agent encodes or is a gene/genome editing component. In some embodiments, the gene/genome editing component is a guide RNA for an RNA-directed nuclease or other nucleic acid editing enzyme, clustered regularly interspaced short palindromic repeat RNA (crisprRNA), a trans-activating clustered regularly interspaced short palindromic repeat RNA (tracrRNA). In some embodiments, the gene/genome editing component is a nucleic acid-encoded enzyme, such as RNA-guided nuclease, a gene or base editing protein, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, a transposase, or a CRISPR nuclease (e.g., Cas9 or Cas 12, etc.). In some embodiments, the gene/genome editing component is DNA to be inserted or that serves as a template in gene or genome editing for example a template for repair of a double-strand break. [00365] In some embodiments comprising multiple agents, the nucleic acid can be multicistronic. In other embodiments comprising multiple agents or components, each agent or component is encoded or contained is a separate nucleic acid species. In some embodiments involving multiple payload nucleic acid species, two or more nucleic acid species are packaged together in a single LNP species. In other embodiments, a subset of the payload nucleic acid species to be delivered, (e.g., a single nucleic acid species) is packaged in one LNP or tLNP species while another subset of the nucleic acid species is packaged in another LNP or tLNP species. The different (t)LNP species can differ by only the payload they contain. The different (t)LNP species can be combined in a single formulation or pharmaceutical composition for administration. Methods of Making an LNP or tLNP [00366] In some aspects, this disclosure provides a method of making a LNP or tLNP comprising mixing of an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of the lipids in proportions disclosed herein. In particular embodiments, the mixing is rapid. [00367] The aqueous solution is buffered at pH of about 3 to about 5, for example, without limitation, with citrate or acetate. In various embodiments, the alcohol can be ethanol, isopropanol, t-butanol, or a combination thereof. In some embodiments, the rapid mixing is accomplished by pumping the two solutions through a T-junction or with an impinging jet mixer. Microfluidic mixing through a staggered herringbone mixer (SHM) or a hydrodynamic mixer (microfluidic hydrodynamic focusing), microfluidic bifurcating mixers, and microfluidic baffle mixers can also be used. After the LNPs are formed they are diluted with buffer, for example phosphate, HEPES, or Tris, in a pH range of about 6 to about 8.5 to reduce the alcohol (ethanol) concentration, The diluted LNPs are purified either by dialysis or ultrafiltration or diafiltration using tangential flow filtration (TFF) against a buffer in a pH range of about 6 to about 8.5 (for example, phosphate, HEPES, or Tris) to remove the alcohol. Alternatively, one can use size exclusion chromatography. Once the alcohol is completely removed the buffer is exchanged with like buffer containing a cryoprotectant (for example, glycerol or a sugar such as sucrose, trehalose, or mannose). The LNPs are concentrated to a desired concentrated, followed by 0.2 µm filtration through, for example, a polyethersulfone (PES) or modified PES filter and filled into glass vials, stoppered, capped, and stored frozen. In alternative embodiments, a lyoprotectant is used and the LNP lyophilized for storage instead of as a frozen liquid. Further methodologies for making LNP can be found, for example, in U.S. Patent Application Publication Nos. US20200297634, US20130115274, and International Patent Application Publication No. WO2017/048770, each of which is incorporated by reference for all that they teach about the production of LNP. [00368] Some aspects are a method of making a tLNP comprising rapid mixing of an aqueous solution of a nucleic acid (or other negatively charged payload) and an alcoholic solution of the lipids as disclosed for LNP. In some embodiments, the lipid mixture includes functionalized PEG-lipid, for later conjugation to a targeting moiety. As used herein, functionalized PEG-lipid refers to a PEG-lipid in which the PEG moiety has been derivatized with a chemically reactive group (such as, maleimide, N-hydroxysuccinimide (NHS) ester, Cys, azide, alkyne, and the like) that can be used for conjugating a targeting moiety to the PEG-lipid, and thus, to the LNP comprising the PEG-lipid. In other embodiments, the functionalized PEG-lipid is inserted into and LNP subsequent to initial formation of an LNP from other components. In either type of embodiment, the targeting moiety is conjugated to functionalized PEG-lipid after the functionalized PEG-lipid containing LNP is formed. Protocols for conjugation can be found, for example, in Parhiz et al.2018, J. Controlled Release 291:106-115, and Tombacz et al., 2021, Molecular Therapy 29(11):3293-3304, each of which is incorporated by reference for all that it teaches about conjugation of PEG- lipids to binding moieties. Alternatively, the targeting moiety can be conjugated to the PEG- lipid prior to insertion into pre-formed LNP. [00369] In certain embodiments of the preparation methods of tLNP, the method comprises: i). forming an initial LNP by mixing all components of the tLNP, in proportions disclosed herein, except for the one or more functionalized PEG-lipids and the one or more targeting moieties; ii). forming a pre-conjugation tLNP by mixing the initial LNP with the one or more functionalized PEG-lipids; and iii). forming the tLNP by conjugating the pre-conjugation tLNP with the one or more targeting moieties. [00370] In certain embodiments of the preparation methods of tLNP, the method comprises: i). forming a pre-conjugation tLNP by mixing all components of the tLNP, in proportions disclosed herein, including the one or more functionalized PEG-lipids, except for the one or more targeting moieties; and ii). forming the tLNP by conjugating the pre-conjugation tLNP with the one or more targeting moieties. [00371] In certain embodiments of the preparation methods of tLNP, the method comprises: i). forming one or more conjugated functionalized PEG-lipids by conjugating the one or more functionalized PEG-lipids with the one or more targeting moieties; and ii) forming the tLNP by mixing all components of the tLNP, including the one or more conjugated functionalized PEG-lipids. [00372] In certain embodiments of the preparation methods of tLNP, the method comprises: i). forming one or more conjugated functionalized PEG-lipids by conjugating the one or more functionalized PEG-lipids with the one or more targeting moieties; ii) forming an LNP by mixing all components of the tLNP, except the one or more conjugated functionalized PEG-lipids; and iii) forming the tLNP by mixing the initial LNP with the one or more conjugated functionalized PEG-lipids. [00373] After the conjugation the tLNPs are purified by dialysis, tangential flow filtration, or size exclusion chromatography, and stored, as disclosed above for LNPs. [00374] The encapsulation efficiency of the nucleic acid by the LNP or tLNP is typically determined with a nucleic acid binding fluorescent dye added to intact and lysed aliquots of the final LNP or tLNP preparation to determine the amounts of unencapsulated and total nucleic acid, respectively. Encapsulation efficiency is typically expressed as a percentage and calculated as 100 x (T-U)/T where T is the total amount of nucleic acid and U is the amount of unencapsulated nucleic acid. In various embodiments, the encapsulation efficiency is ≥80%, ≥85%, ≥90%, or ≥95%. Methods of Delivering a Payload into a Cell [00375] In other aspects, disclosed herein are methods of delivering a nucleic acid (or other negatively charged payload) into a cell comprising contacting the cell with LNP or tLNP disclosed herein. Accordingly, each of the herein disclosed genera, subgenera, and or species of LNP or tLNP disclosed herein including those based on the inclusion or exclusion of particular lipids, particular lipid compositions, particular payloads, and/or particular targeting moieties can be used in defining the scope of the methods of delivering a payload to a cell. In some embodiments, the contacting takes place ex vivo. In some embodiments, the contacting takes place extracorporeally. In some embodiments, the contacting takes place in vivo. In various embodiments, contacting in vivo can be accomplished through any appropriate route of administration. In some embodiments, an LNP or tLNP is contacted with target cells in vivo, by systemic or local administration. In some embodiments, the in vivo contacting comprises intravenous, intramuscular, subcutaneous, intralesional, intranodal or intralymphatic administration. In further instances, transfection of hepatocytes is reduced as compared to tLNPs comprising a conventional, prior art ionizable cationic lipid, such as ALC- 0315. In some embodiments, an LNP or tLNP is administered 1-3 times a week for 1, 2, 3, or 4 weeks. In some embodiments, toxicity is confined (or largely confined) to grades of 0 or 1 or 2, as discussed above. Advantageous compact administration regimens for tLNPs, for example, comprising one or more cycles of 2 or 3 administrations at 72 hour intervals, are described in U.S. Provisional Patent Application Number 63/556,735, filed February 22, 2024, U.S. Provisional Patent Application Number 63/708,513, filed October 17, 2024, U.S. Provisional Patent Application Number 63/721,154, filed November 15, 2024, and PCT patent application No. PCT/US2025/017092, filed February 24, 2025, which administration regimens are incorporated by reference in their entirety. [00376] The herein disclosed LNP and tLNP compositions and formulations have reduced toxicity as compared to widely used prior LNP compositions such as those containing ALC- 0315. In various embodiments the toxicity can be described as an observable toxicity, a substantial toxicity, a severe toxicity, or an acceptable toxicity, or a dose-limiting toxicity (such as but not limited to a maximum tolerated dose (MTD)). By an observable toxicity it is meant that while a change is observed the effect is negligible or mild. By substantial toxicity it is meant that there is a negative impact on the patient’s overall health or quality of life. In some instances, a substantial toxicity can be mitigated or resolved with other ongoing medical intervention. By a severe toxicity it is meant that the effect requires acute medical intervention and/or dose reduction or suspension of treatment. The acceptability of a toxicity will be influenced by the particular disease being treated and its severity and the availability of mitigating medical intervention. In some embodiments, toxicity is confined (or largely confined) to an observable toxicity. In some embodiments, toxicity is confined (or largely confined) to grades of 0 or 1 or 2. [00377] In some embodiments, the payload is a nucleic acid and the method of delivering is a method of transfecting. In some embodiments, the nucleic acid payload comprises an mRNA, circular RNA, self-amplifying RNA, or guide RNA. Nucleic acid structures and especially mRNA structures, as well as individual RNA molecules encoding particular polypeptides, that are well-adapted to delivery by LNP or tLNP are disclosed in U.S. Provisional Patent Application Number.63/595,753 filed November 2, 2023, U.S. Provisional Patent Application Number.63/611,092 filed December 15, 2023, U.S. Provisional Patent Application Number 63/654,928, filed May 31, 2024, U.S. Provisional Patent Application Number 63/708,529, filed October 17, 2024, and International Patent Application No. PCT/US2024/054033, filed November 1, 2024, each of which is incorporated by reference for all that it teaches about nucleic acid payloads for in vivo transfection and their design. [00378] In some embodiments, the payload comprises a nucleic acid encoding an immune receptor or immune cell engager and the method of delivering is also a method of reprogramming an immune cell. In some embodiments, the payload comprises a nucleic acid that encodes, or is, a BRM and the method of delivering is also a method of providing a conditioning agent. In various embodiments, the BRM or conditioning agent is a gamma chain receptor cytokine such as IL-2, IL-7, IL-15, IL-15/15Ralpha, IL-21; an immune modulating (also known as pan activating) cytokine such as IL-12, IL-18; an immune checkpoint inhibitor such as an antagonist of cytotoxic T-lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), program cell death ligand 1 (PD-L1), T- cell immunoglobulin and mucin-domain-containing-3 (Tim-3), lymphocyte activation gene 3 (LAG-3) or indoleamine 2,3-dioxygenase (IDO) or agonists of 4-immunoglobulin and BB cell surface glycoprotein (4-1BB), OX40 or inducible costimulator (ICOS); a chemokine such as RANTES, IP10, MIG; or another BRM such as Flt3, GM-CSF, and G-CSF. [00379] In some embodiments, the payload comprises a nucleic acid encoding a gene/genome editing enzyme and/or a guide RNA or other component of a gene/genome editing system and the method of delivering is also a method of reprogramming a cell. In some instances, the cell is an immune cell. In some instances, the cell is an HSC. In some instances, the cell is an MSC. [00380] In certain embodiments comprising delivering the payload into an immune cell, the binding moiety binds to a lymphocyte surface molecule or a monocyte surface molecule. Lymphocyte surface molecules include CD2, CD3, CD4, CD5, CD7, CD8, CD28, 4-1BB (CD137), CD166, CTLA-4, OX40, PD-1, GITR, LAG-3, TIM-3, CD25, low affinity IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, and IL-21 receptor. Monocyte surface molecules include CD5, CD14, CD16a, CD32, CD40, CD11b (Mac-1), CD64, DEC205, CD68, and TREM2. Exemplary antibodies that can provide antigen binding domains to bind these surface molecules are disclosed herein. Such antibodies, individually and collectively, constitute means for binding to an immune cell (or leukocyte) – or to a lymphocyte or monocyte, as indicated. [00381] In certain embodiments comprising delivering the payload into a stem cell, the binding moiety binds to a HSC surface molecule or a MSC surface molecule. HSC surface molecules include CD117, CD34, CD44, CD90 (Thy1), CD105, CD133, BMPR2, and Sca-1. MSC surface molecules include CD70, CD105, CD73, Stro-1, SSEA-4, CD271, CD146, GD2, SSEA-3, SUSD2, Stro-4, MSCA-1, CD56, CD200, PODXL, CD13, CD29, CD44, and CD10. Exemplary antibodies that can provide antigen binding domains to bind these surface molecules are disclosed herein above. Such antibodies, individually and collectively, constitute means for binding to a stem cell – or to an HSC or MSC, as indicated. Methods of Treatment [00382] In certain aspects, this disclosure provides methods of treating a disease or disorder comprising administering a tLNP (of LNP) of this disclosure to a subject in need thereof. Each of the herein disclosed genera, subgenera, and or species of LNP or tLNP disclosed herein including those based on the inclusion or exclusion of particular lipids, particular lipid compositions, particular payloads, and/or particular targeting moieties can be used in defining the scope of the methods of treatment. [00383] In some embodiments, a subject is a human. In some embodiments, a tLNP is administered systemically. In some embodiments, a tLNP is administered by intravenous or subcutaneous infusion or injection. In some embodiments, a tLNP is administered locally. In some embodiments, a tLNP is administered by intraperitoneal or intralesional infusion injection. [00384] In further embodiments, a tLNP can be administered in combination with the standard of care for a particular indication, such as corticosteroids (e.g., prednisone) for management of myositis or lupus nephritis. In certain cases, myositis is also treated with methotrexate, which can be combined with immunosuppressive agents (e.g., azathioprine, mycophenolate mofetil, tacrolimus), which are usually required in addition to corticosteroids. For membranous nephropathy, cyclical steroids and cyclophosphamide might be used in combination with tLNPs of this disclosure. In other cases, an anti-IL-6, such as tocilizumab, can also be used as a pretreatment or in combination with tLNPs of this disclosure. These combinations can be administered concurrently or sequentially. [00385] In some embodiments, the disease or disorder is an autoimmune disease. Examples of autoimmune disease include, without limitation, myocarditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, fibrosing alveolitis, multiple sclerosis, rheumatic fever, polyglandular syndromes, agranulocytosis, autoimmune hemolytic anemias, bullous pemphigoid, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, allergic responses, insulin-resistant diabetes, psoriasis, diabetes mellitus, Addison’s disease, Grave’s disease, diabetes, endometriosis, celiac disease, Crohn’s disease, Henoch-Schonlein purpura, ulcerative colitis, Goodpasture's syndrome, thromboangitisubiterans, Sjögren's syndrome, aplastic anemia, rheumatoid arthritis, sarcoidosis, scleritis, a T cell-mediated autoimmunity or a B cell-mediated autoimmunity, a B cell-mediated (antibody-mediated) autoimmune disease, necrotizing myopathy, chronic inflammatory demyelinating polyneuropathy (CIDP), neuromyelitis optica (NMO) myositis, neuromyelitis optica spectrum disorders, pemphigus vulgaris, systemic sclerosis, antisynthetase syndrome (idiopathic inflammatory myopathy), lupus nephritis, membranous nephropathy, Fanconi anemia, and vasculitis. [00386] In some embodiments, the autoimmune disease is a T cell-mediated autoimmunity or a B cell-mediated autoimmunity. In some instances, the B cell-mediated autoimmune disease is myositis (such as anti-synthetase myositis), lupus nephritis, membranous nephropathy, systemic lupus erythematosus, anti-neutrophilic cytoplasmic antibody (ANCA) vasculitis, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, pemphigus vulgaris, rheumatoid arthritis, dermatomyositis, immune mediated necrotizing myopathy (IMNM), anti-synthetase syndrome, polymyositis, systemic sclerosis, diffuse cutaneous systemic sclerosis, limited cutaneous systemic sclerosis, anti-synthetase syndrome (idiopathic inflammatory myopathy), stiff person syndrome, myeloid oligodendrocyte glycoprotein autoantibody associated disease (MOGAD), amyloid light- chain amyloidosis, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary progressive multiple sclerosis, primary progressive multiple sclerosis, non-active secondary progressive multiple sclerosis, Sjörgen’s syndrome, IgA nephropathy, IgG4-related disease, or Fanconi anemia. In certain embodiments, the B cell-mediated autoimmune disease is myositis, lupus nephritis, membranous neuropathy, scleroderma, systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris. In certain embodiments, the B cell-mediated autoimmune disease is myositis, lupus nephritis, membranous neuropathy, or scleroderma. In certain embodiments, the B cell- mediated autoimmune disease is myositis. In some instances, the myositis is anti-synthetase myositis. In certain embodiments, the B cell-mediated autoimmune disease is systemic lupus erythematosus, myasthenia gravis, ANCA vasculitis, multiple sclerosis, or pemphigus vulgaris. [00387] In some embodiments, the disease or disorder is rejection of an allogeneic organ or tissue graft. Pre-existing antibodies and/or B cells, in their role as antigen presenting cells, can facilitate rapid immune rejection through known mechanisms hence depleting a large number of B cells can help prevent allograft rejection. Similarly, B cell depletion can be used in the management of graft-versus-host disease. In some embodiments, the disease or disorder is a graft-versus-host disease. [00388] In some embodiments, the disease or disorder is a cancer. Examples of cancers include, without limitation, carcinomas, sarcomas, and hematologic cancers. In some embodiments, the hematologic cancer is a lymphoma, leukemia, or myeloma. In some instances, the hematologic cancer is a B lineage or T lineage cancer. In some instances, the B lineage cancer is multiple myeloma, diffuse large B cell lymphoma, acute myeloid leukemia, Mantle Cell lymphoma, follicular lymphoma, B acute lymphoblastic leukemia, chronic lymphocytic leukemia, or myelodysplastic syndrome. In some embodiments, the cancer is a sarcoma. In some embodiments, the cancer is a carcinoma, such as breast cancer, colon cancer, ovarian cancer, lung cancer, testicular cancer, or pancreatic cancer. In some embodiments, the cancer is melanoma. [00389] Some embodiments are methods of B cell depletion utilizing a CAR or other reprogramming agent with specificity for a B cell antigen. In such context, the term “B cell antigen” can refer to any antigen expressed by cell in the B cell lineage from pro-B cells through plasma cells. Exemplary B cell antigens include CD19, CD20, CD22, BCMA, GPRC5D, and FCRL5, as well as antigens noted to be associated with hematologic cancers of the B cell lineage. In addition to the treatment of B cell cancers, B cell-mediated autoimmunity, allotransplant rejection, GVHD, and the like as disclosed above, B cell depletion blunt induction of anti-drug antibodies as an additional feature of such treatments or as an adjunct to other therapies. U.S. Provisional Patent Application Number 63/556,735, filed February 22, 2024, U.S. Provisional Patent Application Number 63/708,513, filed October 17, 2024, U.S. Provisional Patent Application Number 63/721,154, filed November 15, 2024, and PCT patent application No. PCT/US2025/017092, filed February 24, 2025, describes such uses and is incorporated by reference in its entirety. [00390] In some embodiments, the disease or disorder is a genetic disease or disorder such as a monogenic genetic disease. In some instances, the genetic disease or disorder is a hemoglobinopathy, for example, sickle cell disease or β-thalassemia. [00391] In some embodiments, the disease or disorder is a fibrotic disease or disorder. In some instances, the fibrotic disease is cardiac fibrosis, arthritis, idiopathic pulmonary fibrosis, and nonalcoholic steatohepatitis (also known as metabolic dysfunction-associated steatohepatitis). In other instances, the disorder involves tumor-associated fibroblasts. [00392] In some embodiments, a tLNP of this disclosure comprises a nucleic acid encoding a chimeric antigen receptor (CAR). The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. In some embodiments, a nucleic acid encoding a CAR refers to one or more nucleic acid species encoding one or more CARs; for example, a single or multiple species of nucleic acid encoding a single CAR species, or multiple species of nucleic acid encoding multiple CAR species. In some instances, these multiple CAR species have a same specificity while in other instances they have multiple specificities. In some embodiments, a CAR of this disclosure is multispecific, for example, bispecific, comprising multiple antigen binding moieties each specific for separate antigens. For example, the CAR in LCAR-AIO targets three antigens — CD19, CD20 and CD22 (see, Blood (2021) 138 (Supplement 1): 1700). In some embodiments, a CAR can comprise an extracellular binding domain that specifically binds a target antigen, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, a CAR can further comprise one or more additional elements, including one or more signal peptides, one or more extracellular hinge domains, or one or more intracellular costimulatory domains. Domains can be directly adjacent to one another, or there can be one or more amino acids linking the domains. The signal peptide can be derived from an antibody, a TCR, CD8 or other type 1 membrane proteins, preferably a protein expressed in a T or other immune cell. The transmembrane domain can be one associated with any of the potential intracellular domains or from another type 1 membrane protein, such as TCR alpha, beta, or zeta chain, CD3 epsilon, CD4, CD8, or CD28, amongst other possibilities known in the art. The transmembrane domain can further comprise a hinge domain located between the extracellular binding domain and the hydrophobic membrane-spanning region of the transmembrane domain. In some but not all embodiments, the hinge domain and transmembrane domain are contiguous sequences in the same source protein. In some instances, the hinge and membrane-spanning domains are derived from CD28. In other instances, the hinge and membrane-spanning domains are derived from CD8α. The intracellular signaling domain can be derived from the CD3 zeta chain, DAP10, DAP12, FcγRIII, FcsRI, or an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic domain, amongst other possibilities known in the art. The intracellular costimulatory domain can be derived from CD27, CD28, 4-1BB, OX40, or ICOS, amongst other possibilities known in the art. [00393] In certain embodiments, CARs are used to treat a disease or condition associated with a target cell that expresses the antigen targeted by the CAR. For example, in some embodiments, an anti-CD19 or anti-CD20 CAR can be used to target and treat B cell malignancies or B cell-mediated autoimmune conditions or diseases (e.g., having an immune cell targeting moiety, such as an anti-CD8 antibody). In other embodiments, an anti- FAP CAR can be used to target and treat solid tumors or fibrosis (e.g., cardiac fibrosis, cancer-associated fibroblasts), which can also have an immune cell targeting moiety, such as an anti-CD8 antibody. Examples of CARs that can be used in accordance with the embodiments described herein include to those disclosed in US 7,446,190, US 9,328,156, US 11,248,058, US20190321404, WO2019119822, WO2019159193, WO2020011706, WO2022125837, and WO2024086190 (anti-CD19), US 10,287,35 (anti-CD19), US 10,442,867 and US2021/0363245 (anti-CD19 and anti-CD20), US 10,543,263 (anti-CD22), WO2016149578 (anti-CD19 and anti-CD22), US 10,316,101, US 11,623,961 WO2015052538, WO2016166630, WO2017130223, WO2017173256, WO2019085102, WO2019241426, WO2020065330, WO2020038146, WO2020190737, WO2021091945 (anti-BCMA), WO2016130598 (anti-BCMA and syndecan-1), US 10,426,797 (anti-CD33), US 10,844,128 (anti-CD123), US 10,428,141, US 10,752,684, US 11,723,925, WO2016187216, WO2017156479, WO2018197675, WO2020014366, and WO2020198531 (anti-ROR1), WO2022247756, WO2020148677, WO2020092854, & US20230331872 (anti- GPRC5D), WO2016090337, WO2022263855, & WO2024047558 (anti-FCRL5), and US2021/0087295 (anti-FAP), each of which is incorporated by reference for all that it teaches about CAR structure and function generically and with respect to the CAR’s antigenic specificity and target indications to the extent that it is not inconsistent with this disclosure. Each CAR constitutes means for targeting an immune cell, for example, a T cell, to the indicated antigen. [00394] Exemplary target antigens against which a CAR, TCR, or ICE can have specificity include, but are not limited to, B cell maturation agent (BCMA)† , CA9† , CD4† , CD5† , CD19*† , CD20 (MS4A1)*† , CD22*† , FCRL5† , GPRC5D† , CD23*† , CD30 (TNFRSF8)*† , CD33*† , CD38*† , CD44* , CD70*† , CD133 , CD174, CD274 (PD-L1)*† , CD276 (B7-H3)† , CEACAM5*† , CLL1 , CSPG4* , EGFR*† , EGFRvIII*, EPCAM*† , EPHA2* , ERBB2* , FAP*† , FOLH1, FOLR1*† , GD2*† , GPC3*† , GPNMB* , IL1RAP† , IL3RA* , (mesothelin)* (SDC1)* , CD319 (SLAMF7)*† , CD248 (TEM1) , ULBP1, ULBP2, and G-protein coupled receptor family C group 5 member D (GPRC5D)† (associated with leukemias); CD319 (SLAMF7)*† , CD38*† , CD138† , GPRC5D† , CD267 (TACI) , and BCMA† (associated with myelomas); and GD2*† , GPC3*† , HER2*† , EGFR*† , EGFRvIII*, CD276 (B7H3)† , PSMA*† , PSCA , CAIX (CA9)† , CD171 (L1-CAM)* , CEA* , CSPG4* , EPHA2* , FAP*† , LRRC15† , FOLR1*† , IL-13Rα*† , Mesothelin*† , MUC1*† , MUC16*† , TROP2*† , claudin 18.2† , and ROR1† (associated with solid tumors). Well-known TCR targets include NYESO-1, SSX2, PRAME, lineage specific antigens tyrosinase, PSMA, and Melan A/MART-1, and cancer testes antigens MAGE, GAGE, and LAGE. (* indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in US Patent 11,326,182B2 Table 9 or 10. † indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in Wilkinson & Hale, 2022. Both references cited and incorporated by reference above. indicates that exemplary antibodies with the indicated specificity from which a binding moiety could be derived can be found in the Therapeutic Antibody Database (TABS) at tabs.craic.com. Other suitable antibodies can be found in Appendix A, or in International Patent Publication No. WO2024040195A1, filed August 17, 2023, which is incorporated herein by reference for all that it teaches about individual antibodies and the antigens they bind. Many of these target antigens are themselves receptors that could bind to their ligand if expressed on an immune cell. Accordingly, in some embodiments, the extracellular binding domain of the CAR comprises a ligand of a receptor expressed on the target cell. In still further embodiments, the extracellular binding domain of the CAR comprises a ligand binding domain of a receptor for a ligand expressed on the target cell. The advantages of the aspects and embodiments disclosed herein are independent of the specificity of the binding moiety. As such, the disclosed aspects and embodiments are generally agnostic to binding specificity. In certain embodiments, a particular binding specificity can be required. [00395] In some embodiments, the tLNP comprises a nucleic acid encoding an anti-CD19 chimeric antigen receptor (CAR). In some embodiments, the nucleic acid comprises mRNA. Examples of anti-CD19 CARs include those incorporating a CD19 binding moiety derived from the human antibody 47G4 or the mouse antibody FMC63. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun.34(16-17):1157-1165 (1997) and PCT Application Publication Nos. WO 2018/213337 and WO 2015/187528, the entire contents of each of which are incorporated by reference herein for all that they teach about anti-CD19 CARs and their use. CAR based on 47G4 are disclosed in United States Patent No. 10,287,350 which is incorporated by reference herein for all that it teaches about anti-CD19 CARs and their use. In some instances, the anti-CD19 CAR is the CAR found in tisagenlecleucel, lisocabtagene maraleucel, axicabtagene ciloleucel, or brexucabtagene autoleucel. Each of these CARs constitutes means for targeting an immune cell, for example, a T cell, to CD19. The entire contents of each of foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, and activity of anti-CD19 CARs. In any of the aforementioned tLNP embodiments, certain embodiments include tLNPs encapsulating a CD19 CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody. [00396] In some embodiments, the tLNP comprises a nucleic acid encoding an anti-CD20 chimeric antigen receptor (CAR). CD20 is an antigen found on the surface of B cells as early as the pro-B phase and progressively at increasing levels until B cell maturity, as well as on the cells of most B-cell neoplasms. CD20 positive cells are also sometimes found in cases of Hodgkin's disease, myeloma, and thymoma. In some embodiments, the nucleic acid comprises mRNA. Examples of anti-CD20 CARs include those incorporating a CD20 binding moiety derived from an antibody specific to CD20, including, for example, Leu16, IF5, 1.5.3, rituximab, obinutuzumab, ibritumomab, ofatumumab, tositumumab, odronextamab, veltuzumab, ublituximab, and ocrelizumab. In some embodiments, the anti-CD20 CAR is derived from a CAR specific to CD20, including, for example, MB-106 (Fred Hutchinson Cancer Research Center, see Shadman et al., Blood 134(Suppl.1):3235 (2019)) UCART20 (Cellectis, www.cellbiomedgroup.com), or C-CAR066 (Cellular Biomedicine Group, see Liang et al., J. Clin. Oncol.39(15) suppl:2508 (2021)). In some embodiments, the extracellular binding domain of the anti-CD20 CAR comprises an scFv derived from the Leu16 monoclonal antibody, which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of Leu16 connected by a linker. See Wu et al., Protein Engineering.14(12):1025-1033 (2001). Each of these CARs constitutes means for targeting an immune cell, for example, a T cell, to CD20.The entire contents of each of foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, and activity of anti-CD20 CARs. In any of the aforementioned tLNP embodiments, certain embodiments include tLNPs encapsulating a CD20 CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody. [00397] In some embodiments, the tLNP comprises a nucleic acid encoding an anti- BCMA chimeric antigen receptor (CAR). BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and non- Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the nucleic acid comprises mRNA. Examples of anti-BCMA CARs include those incorporating a BCMA binding moiety derived from C11D5.3, a Mouse monoclonal antibody as described in Carpenter et al., Clin. Cancer Res.19(8):2048-2060 (2013). See also PCT Application Publication No. WO 2010/104949. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from another Mouse monoclonal antibody, C12A3.2, as described in Carpenter et al., Clin. Cancer Res.19(8):2048-2060 (2013) and PCT Application Publication No. WO2010104949. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from a Mouse monoclonal antibody with high specificity to human BCMA, referred to as BB2121 in Friedman et al., Hum. Gene Ther.29(5):585-601 (2018). See also, PCT Application Publication No. WO2012163805. In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oncol.11(1):141 (2018), also referred to as LCAR-B38M. See also, PCT Application Publication No. WO 2018/028647. In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et al., Nat. Commun. 11(1):283 (2020), also referred to as FHVH33. See also, PCT Application Publication No. WO 2019/006072. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv derived from CT103A (or CAR0085) as described in U.S. Patent No.11,026,975 B2. Further anti-BCMA CARs are disclosed in U.S. Application Publication Nos.2020/0246381 and 2020/0339699. Further anti-BCMA CARs include Allo-605 (described in U.S. Patent Publication No.20200261503), CT053 (described in U.S. Patent No. US11,525,006), Descartes-08 (described in U.S. Patent No.10,934,337), LCAR-B38M (described in U.S. Patent No.10,934,363), PersonGen anti-BCMA CAR (described in CN114763383), Pregene Bio anti-BCMA CAR (described in U.S. Patent Publication No. US20220218746), the CAR in ciltacabtagene autoleucel (binding moiety described in US20170051068), and the CAR in idecabtagene vicleucel (described in U.S. Patent No. 10,383,929). Each of these CARs constitutes means for targeting an immune cell, for example, a T cell, to BCMA. Further antibodies comprising an anti-BCMA antigen binding domains that can be used in construction a CAR include AMG224 (described with other anti- BCMA antibodies in U.S. Patent No.9,243,058), EMB-06 (described with other anti-BCMA antibodies in U.S. Patent Publication No. US20230002489), HPN217(described in U.S. Patent No.11,136,403), MEDI2228 (described in U.S. Patent No.10,988,546), REGN5459 (described in U.S. Patent No.11,384,153), SAR445514 (described in U.S. Patent Publication No.20240034816), SEA-BCMA (described in U.S. Patent No.11,078,291), TNB- 383B (described in U.S. Patent No.11,505,606), TQB2934 (described in U.S. Patent Publication No.20230193292), WV078 (described in U.S. Patent No.11,492,409), alnuctamab (described in U.S. Patent No.10,683,369), belantamab (described in U.S. Patent No.9,273,141), elranatamab (described in U.S. Patent No.11,814,435), ispectamab (described in U.S. Patent Publication No.20210130483), linvoseltamab (described in U.S. Patent No.11,919,965), pavurutamab (described in U.S. Patent No.11,419,933), and teclistamab (described in U.S. Patent No.10,072,088). The entire contents of each of foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, and activity of anti-BCMA CARs and anti-BCMA antibodies that can provide an antigen binding domain for a CAR or immune cell engager. In any of the aforementioned tLNP embodiments, certain embodiments include tLNPs encapsulating a BCMA CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti- CD8 antibody. [00398] In some embodiments, the tLNP comprises a nucleic acid encoding an anti- GPRC5D chimeric antigen receptor (CAR). GPRC5D is a G protein-coupled receptor without known ligands and of unclear function in human tissue. However, this receptor is expressed in myeloma cell lines and in bone marrow plasma cells from patients with multiple myeloma. GPRC5D has been identified as an immunotherapeutic target in multiple myeloma and Hodgkin lymphomas. Examples of anti-GPRC5D CARs include those incorporating a GPRC5D binding moiety such as MCARH109 (Mailankody et al., N Engl J Med.387(13): 1196-1206 (2022)), BMS-986393, or OriCAR-017 (Rodriguez-Otero et al., Blood Cancer J. 14(1): 24 (2024)). Examples of anti-GPRC5D CARs include those incorporating a GPRC5D binding moiety derived from an antibody specific to GPRC5D, for example, talquetamab (Pillarisetti et al., Blood 135:1232-43 (2020)), or forimtamig. In some embodiments, the extracellular binding domain of the anti-GPRC5D CAR comprises an scFv derived from a 6D9 Mouse antibody with specificity to human GPRC5D (see creative-biolabs.com/car-t/anti- gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm). In some embodiments, the extracellular binding domain of the GPRC5D CAR comprises an scFv of anti-GPRC5D antibody linked to 4-1BB or CD28 costimulatory domain and CD3ζ signaling domain as described in Mailankody et al., N Engl J Med.387(13): 1196-1206 (2022); creative-biolabs.com/car-t/anti- gprc5d-6d9-h-41bb-cd3-car-pcdcar1-26380.htm; and Rodriguez-Otero et al., Blood Cancer J.14(1): 24 (2024). The entire contents of each of foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, and activity of anti-GPRC5D CARs and anti-GPRC5D antibodies that can provide an antigen binding domain for a CAR or immune cell engager, and each example constitutes a means for binding GPRC5D. In any of the aforementioned tLNP embodiments, certain embodiments include tLNPs encapsulating an anti-GPRC5D CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody. [00399] In some embodiments, the tLNP comprises a nucleic acid encoding an anti- FCRL5 chimeric antigen receptor (CAR). FCRL5 (Fc receptor-like 5), also known as FCRH5, BXMAS1, CD307, CD307E, and IRTA2, is a protein marker expressed on the surface of plasma cells in patients with multiple myeloma. Furthermore, contact with FCRL5 stimulates B-cell proliferation; thus, FCRL5 has been identified as an immunotherapeutic target for this disease. Examples of anti-FCRL5 CARS include those incorporating an FCRL5 binding moiety, such as those described in WO2016090337, WO2017096120, WO2022263855, and WO2024047558. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises an scFv with specificity to FCRL5, such as ET200-31, ET200-39, ET200-69, ET200-104, ET200-105, ET200-109, or ET200-117. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises an scFv derived from a mouse antibody with specificity to human FCRL5. Such antibodies include 7D11, F25, F56, and F119, as described in Polson et al., Int. Immunol., 18(9): 1363-1373 (2006); Franco et al., J. Immunol. 190(11): 5739-5746 (2013); Ise et al., Clin. Cancer Res.11(1): 87-96 (2005); and Ise et al., Clin. Chem. Lab. Med.44(5): 594-602 (2006), all of which are incorporated by reference herein. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises a binding moiety derived from the antigen binding domain of an anti-FCRL5 antibody or nanobody, including cevostamab, 2A10H7, 307307, 2A10D6, 13G9, 10A8, 509f6, EPR27365-87, EPR26948-19, or EPR26948-67, or as disclosed in WO2016090337, WO2017096120, WO2022263855, or WO2024047558. In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR comprises a binding moiety derived from an antibody-drug conjugate targeting FCRL5, such as those described in Elkins et al., Mol. Cancer Ther.11(10): 2222-2232 (2012). In some embodiments, the extracellular binding domain of the anti-FCRL5 CAR is linked to a costimulatory domain, such as a 4-1BB or CD28 costimulatory domain, and a signaling domain, such as a CD3ζ signaling domain. The entire contents of each of the foregoing references in this paragraph are incorporated by reference for all that they teach about the design, structure, properties, and activity of anti- FCRL5 CARs and anti-FCRL5 antibodies that can provide an antigen binding domain for a CAR or immune cell engager. Each example constitutes a means for binding FCRL5. In any of the aforementioned tLNP embodiments, certain embodiments include tLNPs encapsulating a FCRL5 CAR payload encoded by RNA and having a T cell targeting moiety, such as an anti-CD8 antibody. [00400] In some embodiments, the tLNP comprises nucleic acids encoding one or more CARs that target one or more multiple antigens. In some embodiments, the tLNP comprises distinct mRNAs that are encapsulated together in a single tLNP, with each mRNA encoding one monospecific CAR. For examples, the tLNP can comprise an mRNA encoding an anti- CD19 CAR and an mRNA encoding an anti-CD20 CAR, an mRNA encoding an anti-CD19 CAR and an mRNA encoding an anti-BCMA CAR, an mRNA encoding an anti-GPRC5D CAR and an mRNA encoding an anti-BCMA CAR, or an mRNA encoding an anti-FCRL5 CAR and an mRNA encoding an anti-BCMA CAR. In some embodiments, the tLNP comprises a single mRNA encoding a bicistronic mRNA encoding two monospecific CARs. For examples, the bicistronic mRNA can encode an anti-CD19 CAR and an anti-CD20 CAR, an anti-CD19 CAR and an anti-BCMA CAR, an anti-GPRC5D CAR and an anti-BCMA CAR, or an anti-FCRL5 CAR and an anti-BCMA CAR. In some embodiments, the tLNP comprises a single mRNA encoding an mRNA encoding a multispecific CAR. In some embodiments, the tLNP comprises a single mRNA encoding an mRNA encoding a bispecific CAR. For examples, the mRNA can encode an anti-CD19 and anti-CD20 bispecific CAR, an anti-CD19 and anti-BCMA bispecific CAR, an anti-GPRC5D and anti-BCMA bispecific CAR, or an anti- FCRL5 and anti-BCMA bispecific CAR. In some embodiments, multiple tLNPs can be co- formulated in a combination with each comprising one mRNA encoding one monospecific CAR. For examples, two tLNPs can be co-formulated with one tLNP comprising an mRNA encoding an anti-CD19 CAR and the other tLNP comprising an mRNA encoding an anti- CD20 CAR, one tLNP comprising an mRNA encoding an anti-C19 CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR, one tLNP comprising an mRNA encoding an anti-GPRC5D CAR and the other tLNP comprising an mRNA encoding an anti- BCMA CAR, or one tLNP comprising an mRNA encoding an anti-FCRL5 CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR. In some embodiments, multiple tLNPs can be co-administered in a combination, either simultaneously or sequentially, wherein each comprises one mRNA encoding one monospecific CAR. For examples, two tLNPs can be co-administered in a combination, either simultaneously or sequentially, with one tLNP comprising an mRNA encoding an anti-CD19 CAR and the other tLNP comprising an mRNA encoding an anti-CD20 CAR, one tLNP comprising an mRNA encoding an anti- C19 CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR, one tLNP comprising an mRNA encoding an anti-GPRC5D CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR, or one tLNP comprising an mRNA encoding an anti- FCRL5 CAR and the other tLNP comprising an mRNA encoding an anti-BCMA CAR. The targeting can be mediated by any of the CARs described herein which constitute means for targeting cells expressing the indicated antigen. [00401] Cellular therapy involving the administration of genetically engineered cells to a patient has generally required depleting or ablative conditioning to facilitate engraftment of the engineered cells (for example, T cells or HSC). In the context of in vivo engineering and reprogramming such conditioning would be counterproductive as the conditioning would eliminate the very cells that are to be engineered. Instead, one can utilize activating and/or adjuvant conditioning to increase the number of cells amenable to engineering, to mobilize them to the locus of pathology, to make the locus of pathology (for example, a tumor microenvironment) more susceptible to treatment, to augment the therapeutic effect, etc., as appropriate for the particular disease and primary treatment. Conditioning agents include biological response modifiers (BRMs) that can be delivered directly to a subject or encoded in nucleic acid molecules, including as mRNA, and delivered to a subject using the LNP and tLNP compositions and formulations disclosed herein. [00402] Accordingly, certain aspects are methods of conditioning a subject who receives an engineering agent comprising providing a tLNP comprising a nucleic acid molecule encoding a conditioning agent to the subject prior to, concurrently with, or subsequent to administration of the engineering agent. In various embodiments, an encoded conditioning agent comprises a γ-chain receptor agonist, an inflammatory chemokine, a pan-activating cytokine, an antigen presenting cell activity enhancer, an immune checkpoint inhibitor, or an anti-CCR4 antibody. In some embodiments, the γ-chain receptor cytokine comprises IL-15, IL-2, IL-7, or IL-21. In some embodiments, the immune checkpoint inhibitor comprises an anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-Tim-3, or anti-LAG-3 antibody. In some embodiments, the inflammatory chemokine comprises CCL2, CCL3, CCL4, CCL5, CCL11, CXCL1, CXCL2, CXCL-8, CXCL9, CXCL10, or CXCL11. In some embodiments, the antigen presenting cell activity enhancer comprises Flt-3 ligand, gm-CSF, or IL-18. In some embodiments, a pan-activating cytokine comprises IL-12 of IL 18. In certain embodiments, a conditioning agent comprises a transcription factor, for example, one selected from the group consisting of nuclear factor of activated T-cells (NFAT), NF-κB, T-bet, signal transducer and activator of transcription 4 (STAT4), Blimp-1, c-Jun, and Eomesodermin (Eomes) and the tLNP is targeted to a T cell. In some embodiments, a tLNP encapsulating the nucleic acid-encoded conditioning agent is administered systemically, for example, by intravenous or subcutaneous infusion or injection. In other embodiments, the tLNP is administered locally, for example, by intralesional or intraperitoneal injection or infusion. In some embodiments, nucleic acid molecules encoding the conditioning agent and the engineering agent are encapsulated in the same tLNP while in other embodiments they are encapsulated in separate tLNPs. These two modes of delivery of conditioning agents are described in greater detail in PCT application PCT/US 2023/072426, which is incorporated by reference for all that it teaches about conditioning agents and their delivery of LNPs or tLNPs that is not inconsistent with this disclosure. In some embodiments, the nucleic acid comprises mRNA. [00403] The term “treating” or “treatment” broadly includes any kind of treatment activity, including the mitigation, cure or prevention of disease, or aspect thereof, in man or other animals, or any activity that otherwise affects the structure or any function of the body of man or other animals. Treatment activity includes the administration of the medicaments, dosage forms, and pharmaceutical compositions described herein to a patient, especially according to the various methods of treatment disclosed herein, whether by a healthcare professional, the patient his/herself, or any other person. Treatment activities include the orders, instructions, and advice of healthcare professionals such as physicians, physician’s assistants, nurse practitioners, and the like, that are then acted upon by any other person including other healthcare professionals or the patient him/herself. In some embodiments, the orders, instructions, and advice aspect of treatment activity can also include encouraging, inducing, or mandating that a particular medicament, or combination thereof, be chosen for treatment of a condition - and the medicament is actually used - by approving insurance coverage for the medicament, denying coverage for an alternative medicament, including the medicament on, or excluding an alternative medicament, from a drug formulary, or offering a financial incentive to use the medicament, as might be done by an insurance company or a pharmacy benefits management company, and the like. In some embodiments, treatment activity can also include encouraging, inducing, or mandating that a particular medicament be chosen for treatment of a condition - and the medicament is actually used - by a policy or practice standard as might be established by a hospital, clinic, health maintenance organization, medical practice or physicians’ group, and the like. All such orders, instructions, and advice are to be seen as conditioning receipt of the benefit of the treatment on compliance with the instruction. In some instances, a financial benefit is also received by the patient for compliance with such orders, instructions, and advice. In some instances, a financial benefit is also received by the healthcare professional for compliance with such orders, instructions, and advice. [00404] Some embodiments of these methods of treatment comprise administration of an effective amount of a compound or a composition disclosed herein. Some instances relate to a therapeutically (or prophylactically) effective amount. A therapeutically effective amount is not necessarily a clinically effective amount, that is, while there can be therapeutic benefit as compared to no treatment, a method of treatment may not be equivalent or superior to a standard of care treatment existing at some point in time. Other instances relate to a pharmacologically effective amount, that is an amount or dose that produces an effect that correlates with or is reasonably predictive of therapeutic (or prophylactic) utility. As used herein, the term “therapeutically effective amount” is synonymous with “therapeutically effective dose” and means at least the minimum dose of a compound or composition disclosed herein necessary to achieve the desired therapeutic or prophylactic effect. Similarly, a pharmacologically effective dose means at least the minimum dose of a compound or composition disclosed herein necessary to achieve the desired pharmacologic effect. Some embodiments refer to an amount sufficient to prevent or disrupt a disease process, or to reduce the extent or duration of pathology. Some embodiments refer to a dose sufficient to reduce a symptom associated with the disease or condition being treated. [00405] The following examples are intended to illustrate various embodiments. As such, the specific embodiments discussed are not to be constructed as limitations on the scope of this disclosure. It is apparent to one skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
EXAMPLES Example 1: Synthesis of 5-(Octyloxy)-3-(2-(octyloxy)-2-oxoethyl)-5-oxopentanoic acid: 2 [00406] Step 1: To diacid 1 (J. Am. Chem. Soc. 2010, 132, 15790, 14.0g, 56.91mmol), dissolved in acetonitrile (140mL) at 25°C under nitrogen, was added in order 1-octanol (14.70g, 113.82mmol), DMAP (7.93g, 56.91mmol) and EDCl (27.32g, 142.28mmol). The mixture was stirred for 18 hours at 25°C, the was diluted with n-heptane (500mL) and the resulting solution was washed with water (200mL). The organic phase was separated silica gel (50g, type ZCX-2, 100-200 mesh) was added and the solvent was removed in vacuo. The dry, reaction impregnated silica gel, was placed atop a column of silica gel (500g, type ZCX- 2, 100-200 mesh) in a Combi-flash apparatus. The column was eluted with a gradient of n- heptane/EtOAc from 100:0 to 95:5. Qualified fractions of the resulting t-butyl ester, of step 1, eluted between n-heptane/EtOAc 98:2 and 97:3 (24.9g, 51.2mmol, 90% yield). [00407] 1H-NMR (300mHz, CDCl3): ^ = 4.07 (t, J = 6.6 Hz, 4H), 2.62 (m, 1H), 2.47 (d, J = 6.6 Hz, 4H), 2.38 (d, J = 6.6 Hz, 2H), 1.62 (m, 4H), 1.45 (s, 9H), 1.25-1.40 (20H), 0.89 (t, J = 6.9 Hz, 6H); LCMS: RT 1.705min, Calcd. for C23H42O6 (M-t-Bu + 2H+) 415.31. Found 415.30. [00408] Step 2: A solution of the t-butyl ester of step 1 (24.0g, 51.0mmol) in toluene (110 mL) was cooled to 5°C under nitrogen, then CF3CO2H (53.64g, 0.47mol, 36mL) was added dropwise over 30 minutes. The mixture was allowed to warm to room temperature and was stirred at room temperature for 18 hours. The solution was diluted with n-heptane (370mL) and then 10% aq. K2HPO4 solution (500mL) was added, and the mixture was stirred for 10 minutes. The organic phase was separated, and the aqueous phase was extracted with n- heptane (2x250mL). The aqueous phase was acidified by the addition of 1.2M aq. HCl (250mL) and the solution was extracted with n-heptane (500mL). The organic phase was washed with an aqueous methanol solution (1:1, 250mL) and then was dried over anhydrous Na2SO4. Filtration and concentration in vacuo afforded acid 2 (18.0g, 43.4mmol, 85% yield) as a pale-yellow oil. [00409] 1H-NMR (300mHz, CDCl3): ^ = 4.07 (t, J = 6.6 Hz, 4H), 2.77 (m, 1H), 2.55 (d, J = 6.6 Hz, 2H), 2.49 (d, J = 6.6 Hz, 4H), 1.64 (m, 4H), 1.22-1.37 (20H), 0.88 (t, J = 6.9 Hz, 6H); LCMS: RT 1.548min, Calcd. for C23H42O6 (M + H+) 415.31 (base). Found 415.30. Example 2: Synthesis of O'1,O1-(((tert-Butoxycarbonyl)azanediyl)bis(ethane-2,1-diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): 4 [00410] To a solution of 2 (8.08g, 19.51mmol), in acetonitrile (160mL) under nitrogen at 25°C, was added in order diol 3 (2.00g, 9.76mmol), DMAP (1.19g, 9.76mmol) and EDCl (4.68g, 24.39mmol). The resulting solution was allowed to stir for 18h at 25°C, then was concentrated in vacuo. The residue was dissolved in n-heptane(400mL), and the resulting solution was washed with MeOH/10% aq. citric acid (3x100mL, 5:1), water (2x100mL) and dried over anhydrous Na2SO4. Filtration and concentration in vacuo gave 4 (9.00g, 92% yield, 92% purity by HPLC) as a light-yellow oil. [00411] 1H-NMR (300mHz, CDCl3): ^ = 4.19 (m, 4H), 4.07 (t, J = 6.6 Hz, 8H), 3.40 (m, 4H), 2.78 (m, 2H), 2.40-2.57 (12H), 1.63 (m, 8H), 1.47 (s, 9H), 1.23-1.40 (40H), 0.89 (m, 12H); LCMS: RT 1.197min, Calcd. for C55H99NNaO14 (M + Na+) 1020.69 (40). Found 1020.80. Calcd. for C50H92NO12 (M – CO2tBu + 2H+) 898.66. Found 898.80 (base).
Example 3: Synthesis of bis(2-((5-(Octyloxy)-3-(2-(octyloxy)-2-oxoethyl)-5- oxopentanoyl) oxy)ethyl)ammonium trifluoroacetate: 5 [00412] To a solution of 4 (9.00g, 9.02mmol), in dichloromethane (DCM, 45mL) under nitrogen at 25°C, was added CF3CO2H (20.11g, 176mmol, 13.5mL) over a period of 10 minutes. The mixture was allowed to stir for 3 hours at 25°C, then was concentrated in vacuo and the residue was dissolved in n-heptane (400mL). The resulting solution was washed with water (2x200mL) and the organic phase was dried over anhydrous Na2SO4.Filtration and concentration in vacuo provided crude 5 (8.80g, 96% yield, 92% HPLC purity) [00413] 1H-NMR (300mHz, CDCl3): ^ = 4.49 (m, 4H), 4.08 (t, J = 6.6 Hz, 8H), 3.30 (m, 4H), 2.79 (m, 2H), 2.58 (d, J = 6.6 Hz, 4H), 2.48 (d, J = 6.6 Hz, 8H), 1.62 (m, 8H), 1.20-1.37 (40H), 0.89 (m, 12H); LCMS: RT 1.625min, Calcd. for C55H99NNaO14 (M + Na+) 1020.69 (40). Found 1020.80. Calcd. for C50H92NO12898.66. Found 898.70 (base). Example 4: Synthesis of O'1,O1-(((1H-Imidazole-1-carbonyl)azanediyl)bis(ethane-2,1- diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): 6 [00414] To a solution of 6 (8.80g, crude, 92% HPLC purity) in DCM (260mL), under nitrogen at 25°C was added carbonyl diimidazole (CDI, 14.30g, 88.3mmol). The resulting solution was stirred for 18 hours at 25°C then was washed with 1M aq. HCl (3x100mL), brine (2x100mL) and the organic phase was dried over anhydrous Na2SO4. Filtration and concentration in vacuo gave crude 6 as a yellow oil which was dissolved in n-heptane and the solution was washed with a methanol-water solution (4:1, 2x100mL), water (100mL), and dried over anhydrous Na2SO4. Filtration and concentration in vacuo provided crude 8 (8.50g, 87.4% HPLC purity) as a pale-yellow oil. [00415] 1H-NMR (300mHz, CDCl3): ^ =7.99 (s, 1H), 7.33 (brs, 1H), 7.11 (brs, 1H), 4.29 (m, 4H), 4.05 (t, J = 6.9Hz, 8H), 3.76 (m, 4H), 2.52 (m, 2H), 2.42-2.52 (12H), 1.60 (m, 8H), 1.18-1.40 (40H), 0.88 (m, 12H); LCMS: RT 1.719min, Calcd. for C55H93N3O14 (M + H+) 992.7. Found 992.6 (base). Example 5: Synthesis of O'1,O1-(((4-(dimethylamino)butanoyl)azanediyl)bis(ethane-2,1- diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-250 [00416] To a solution of 6 (8.50g crude, 87.5% HCLC purity) in acetonitrile (85mL), cooled to 0°C under nitrogen, was added methyl trifluoromethanesulfonate (MeOTf, 1.55g, 9.43mmol) over a period of 5 minutes. The mixture was stirred for 2 hours at 0°C, then (CH3)3N (trimethyl amine, 1.52g, 25.71mmol) was added, followed by the addition of 2-N,N-dimethylamino- ethanol (3.81g, 42.84mmol) in one portion. The mixture was stirred for 30 minutes at 0°C then was warmed to 50°C and stirring was continued for 18 hours. The mixture was cooled to room temperature and concentrated in vacuo, to give crude CICL-250 as a viscous yellow oil. Crude CICL-250 was dissolved in n-heptane (400mL) and the solution was washed with a methanol- water solution (5:1, 2x100mL). The organic phase was dried over anhydrous Na2SO4, filtered and concentrated in vacuo and the crude CICL-250 was dissolved in acetonitrile and purified by reversed phase flash chromatography (Column: XB-phenyl; mobile phase CH3CN and 0.1% CF3CO2H in water, 50-90% gradient over 18 minutes; detector: UV 200nM) . Qualified fractions were pooled and concentrated in vacuo to remove acetonitrile and the residue was diluted with n-heptane (400mL). The pH of the aq. phase was basified to pH = 8 with 2% aq. Na2CO3 and the organic phase was separated. The organic phase was washed with a methanol-water solution (5:1, 2x100mL), water (200mL), then dried over anhydrous Na2SO4. Filtration and concentration in vacuo yielded CICL-250 (2.18g, 25.1% yield, 97.5% HPLC purity) as a pale-yellow oil. [00417] 1H-NMR (300mHz, CDCl3): ^ =4.15-4.35 (6H), 4.06 (m, 8H), 3.54 (m, 4H), 2.75 (m, 2H), 2.45-2.60 (12H), 2.30 (s, 6H), 1.55-1.65 (8H), 1.25-1.32 (40H), 0.89 (t, J = 6.3 Hz, 12H); LCMS: RT 1.615min, Calcd. for C55H101N2O14 (M + H+) 1013.7. Found 1013.6. Example 6: Synthesis of O'1,O1-((((2-(Dimethylamino)ethoxy)carbonyl) azanediyl) bis(ethane-2,1-diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL- 250 [00418] 3-(Carboxymethyl)pentanedioic acid 1 (Chem. Eur. J.2010, 16, 4037; Org. Chem. Frontiers 2017, 4, 1819) is coupled with 1-octanol, utilizing EDC-HCl and DMAP in CH3CN to give 2. The coupling of diester 2 with diol 3 (CombiBlocks #QB-8577), utilizing EDC-HCl and DMAP in CH3CN, results in 4. Deprotection of the BOC-amine 4, with CF3CO2H in CH2Cl2, provides ammonium trifluoroacetate salt 5. Treatment of 5 with carbonyl-di-imidazole and Et3N in CH2Cl2, gives acyl-imidazole 6. Alkylation of 6 with MeOTf in CH3CN, followed by the reaction of the derived acyl-imidazolium salt with 2- dimethylaminoethanol and (CH3)3N leads to CICL-250. Example 7: Synthesis of O'1,O1-(2-(((2-(dimethylamino)ethoxy)carbonyl) amino)propane-1,3-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-291 [00419] The coupling of diester 2 with diol 7 (Combi-Blocks #QI-4068), utilizing EDC-HCl and DMAP in CH3CN, results in 8. Deprotection of the BOC-amine 8, with CF3CO2H in CH2Cl2, provides ammonium trifluoroacetate salt 9. Treatment of 9 with carbonyl-di- imidazole and Et3N in CH2Cl2, gives acyl-imidazole 10. Alkylation of 10 with MeOTf in CH3CN, followed by the reaction of the derived acyl-imidazolium salt with 2- dimethylaminoethanol and (CH3)3N leads to CICL-291. Example 8: Synthesis of O'1,O1-(2-(2-(2-(dimethylamino)ethoxy)-2-oxoethyl)propane- 1,3-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-292 [00420] The coupling of diester 2 with diol 11 (Org. Proc. Res. Dev.2011, 15, 515), utilizing EDC-HCl and DMAP in CH3CN, results in 12. Deprotection of the t-butyl ester 12, with CF3CO2H in CH2Cl2, provides acid 13. The coupling of acid 13 with N,N- dimethylaminoethanol, using HATU and i-Pr2NET in THF, leads to CICL-292.
Example 9: Synthesis of O'1,O1-(3-((3-(dimethylamino)propanoyl)oxy)pentane-1,5-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-293 [00421] The coupling of diester 2 with diol 14 (Tetrahedron 1987, 43, 45), utilizing EDC- HCl and DMAP in CH3CN, results in 15. Hydrolysis of the THP ether of 15 with PPTS in MeOH, affords alcohol 16. The coupling of alcohol 16 with N,N-dimethyl-^-alanine, using EDC-HCl, DMAP, and Et3N in CH2Cl2, leads to CICL-293.
Example 10: Synthesis of O'1,O1-(3-(3-(dimethylamino)propanamido)pentane-1,5-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-294 [00422] The coupling of diester 2 with diol 17 (Org. Proc. Res. Dev.2009, 13, 428), utilizing EDC-HCl and DMAP in CH3CN, results in 18. Deprotection of the BOC-amine 18, with CF3CO2H in CH2Cl2, provides ammonium trifluoroacetate salt 19. Treatment of 19 with N,N-dimethyl-^-alanine, in the presence of EDC-HCl and DMAP in CH3CN, results in CICL- 294.
Example 11: Synthesis of O'1,O1-(3-(3-(dimethylamino)-N- methylpropanamido)pentane-1,5-diyl) 5,5'-dioctyl bis(3-(2-(octyloxy)-2- oxoethyl)pentanedioate): CICL-295 [00423] The reaction of dimethyl-3-oxopentanedioate 20 with N-methyl benzyl amine in methanol (Synthesis 2011, 2781) produces enamine 21. The reduction of 21 with borane-t- butylamine complex (Org. Proc. Res. Dev.2009, 13, 478) provides amine 22 which leads to amino diol 23 after reduction with LiAlH4 in THF or diethyl ether. Hydrogenolysis (H2, Pd/C) of 23 in the presence of BOC-anhydride then affords N-protected amino diol 24. The coupling of 24 with acid 2 (EDC-HCl, DMAP, Et3N in CH2Cl2, THF, or CH3CN) provides BOC-protected amine 25 which yields the ammonium trifluoroacete salt 26 after BOC removal (CF3CO2H in CH2Cl2 or toluene). Amine salt 26 then couples (EDC-HCl, DMAP, Et3N in CH2Cl2, THF, or CH3CN) with 3-dimethylamino propionic acid to prepare CICL-295. Example 12: Synthesis of O'6,O6-(2-((2-(dimethylamino)ethoxy)carbonyl)-2- methylpropane-1,3-d iyl) 1,1'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)hexanedioate): CICL-296. [00424] The reaction of aldehyde 27 (CombiBlocks #JP-474) with the Wittig reagent 28 (CombiBlocks #QP-4253) results in unsaturated diester 29. A Mukaiyama-Michael addition of 29 with enol silyl ether 30 (Tetrahedron Letters 2002, 43, 7753,) catalyzed by aluminum triflate (J. Org. Chem.1992, 57, 4746), would lead to tri-ester 31. Cleavage of the benzyl esters, with hydrogen and palladium on carbon, provides diacid 32, which when reacted with 1-octanol, utilizing EDC-HCl and DMAP as the coupling conditions, leads to triester 33. Cleavage of the t-butyl ester with CF3CO2H in CH2Cl2 (Synthesis 2005, 11, 1829) affords mono-acid 34. Mono-acid 34 is coupled with diol 35 (J. Am. Chem. Soc.2011, 133, 20288), which affords 36. Hydrogenolytic benzyl ester cleavage (H2, Pd/C) leads to acid 37, which is coupled with N,N-dimethylaminoethanol (EDC-HCl, DMAP) to furnish CICL-296. Example 13: Synthesis of Tetraoctyl 2,2',2'',2'''-((((((2-(dimethylamino)ethoxy)carbonyl) azanediyl)bis(ethane-2,1-diyl))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(azanetriyl)) tetraacetate: CICL-297 [00425] 2,2'-((2-(Benzyloxy)-2-oxoethyl)azanediyl)diacetic acid 38 (WO2018006063 p251), when coupled (EDC-HCl, DMAP) with 1-octanol, provides triester 39. Hydrogenolytic cleavage of the benzyl ester of 39 (H2, Pd/C) results in the formation of diester-acid 41. The coupling of diester 40 with diol 3, utilizing EDC-HCl and DMAP in CH3CN, results in 41. Deprotection of the BOC-amine 41, with CF3CO2H in CH2Cl2, provides ammonium trifluoroacetate salt 42. Treatment of 42 with carbonyl-di-imidazole and Et3N in CH2Cl2, gives acyl-imidazole 43. Alkylation of 43 with MeOTf in CH3CN, followed by the reaction of the derived acyl-imidazolium salt with 2-dimethylaminoethanol and (CH3)3N leads to CICL-297. Example 14: Synthesis of Tetraoctyl 2,2',2'',2'''-((((2-(((2- (dimethylamino)ethoxy)carbonyl) amino)propane-1,3-diyl)bis(oxy))bis(2-oethane-2,1- diyl))bis(azanetriyl))tetraacetate: CICL-298 [00426] The coupling of diester 40 with diol 7, using utilizing EDC-HCl and DMAP in CH3CN, results in 44. Deprotection of the BOC-amine 44, with CF3CO2H in CH2Cl2, provides ammonium trifluoroacetate salt 45. Treatment of 45 with carbonyl-di-imidazole and Et3N in CH2Cl2, gives acyl-imidazole 46. Alkylation of 46 with MeOTf in CH3CN, followed by the reaction of the derived acyl-imidazolium salt with 2-dimethylaminoethanol and (CH3)3N leads to CICL-298. Example 15: Synthesis of Tetraoctyl 2,2',2'',2'''-((((2-(2-(2-(dimethylamino) ethoxy)-2- oxoethyl)propane-1,3-diyl)bis(oxy))bis(2-oxoethane-2,1- diyl))bis(azanetriyl))tetraacetate: CICL-299 [00427] The coupling of diester 40 with diol 11, utilizing EDC-HCl and DMAP in CH3CN, results in 47. Deprotection of the t-butyl ester 47, with CF3CO2H in CH2Cl2, provides acid 48. The coupling of acid 48 with N,N-dimethylaminoethanol, using HATU and i-Pr2NET in THF, leads to CICL-299.
Example 16: Synthesis of Tetraoctyl 2,2',2'',2'''-((((3-((3-(dimethylamino)propanoyl)oxy) pentane-1,5-diyl)bis(oxy))bis(2-oxoethane-2,1-diyl))bis(azanetriyl))tetraacetate: CICL- 300 [00428] The coupling of diester 40 with diol 14, utilizing EDC-HCl and DMAP in CH3CN, produces 49. Hydrolysis of the THP ether of 49 with PPTS in MeOH, affords alcohol 50. The coupling of alcohol 50 with N,N-dimethyl-^-alanine, using EDC-HCl, DMAP, and Et3N in CH2Cl2, leads to CICL-300.
Example 17: Synthesis of Tetraoctyl 2,2',2'',2'''-((((3-(3-(dimethylamino)propanamido) pentane-1,5-diyl)bis(oxy))bis(2-oxoethane-2,1-diyl))bis(azanetriyl))tetraacetate: CICL- 301 [00429] The coupling of diester 40 with diol 17, utilizing EDC-HCl and DMAP in CH3CN, results in 51. Deprotection of the BOC-amine 51, with CF3CO2H in CH2Cl2, provides ammonium trifluoroacetate salt 52. Treatment of 52 with N,N-dimethyl-^-alanine, in the presence of EDC-HCl and DMAP in CH3CN, results in CICL-301.
Example 18: Synthesis of Tetraoctyl 2,2',2'',2'''-((((3-(3-(dimethylamino)-N- methylpropanamido)pentane-1,5-diyl)bis(oxy))bis(2-oxoethane-2,1- diyl))bis(azanetriyl)) tetraacetate: CICL-302 [00430] The coupling of 24 with acid 40 (EDC-HCl, DMAP, Et3N in CH2Cl2, THF, or CH3CN) provides BOC-protected amine 53, which yields the ammonium trifluoroacete salt 54 after BOC removal (CF3CO2H in CH2Cl2 or toluene). Amine salt 54 then couples (EDC- HCl, DMAP, Et3N in CH2Cl2, THF, or CH3CN) with 3-dimethylamino propionic acid to prepare CICL-302.
Example 19: Synthesis of Tetraoctyl 2,2',2'',2'''-((((2-((2- (dimethylamino)ethoxy)carbonyl)-2-methylpropane-1,3-diyl)bis(oxy))bis(3- oxopropane-3,1-diyl))bis(azanetriyl))tetraacetate: CICL-303 [00431] Hydrolysis of the bis-t-butyl esters present in 55 (Bioorg. Med. Chem. Lett.2014, 24, 2855), with CF3CO2H in CH2Cl2, results in diacid 56, which when reacted with 1-octanol, utilizing EDC-HCl and DMAP as the coupling conditions, leads to triester 57. Cleavage of the benzyl ester with hydrogen and Pd/C affords mono-acid 58. Mono-acid 58 is coupled with diol 35 which affords 59. Hydrogenolytic benzyl ester cleavage (H2, Pd/C) leads to acid 60, which is coupled with N,N-dimethylaminoethanol (EDC-HCl, DMAP) to furnish CICL-303.
Example 20: Synthesis of O'1,O1-(((((1-methylazetidin-3-yl)oxy)carbonyl)azanediyl)bis (ethane-2,1-diyl)) 5,5'-dioctyl bis(3-(2-(octyloxy)-2-oxoethyl)pentanedioate): CICL-250- 61 [00432] Acylimidazolide 6, when reacted with MeOTf provides an activated acylimidazolium species. The reaction of the activated acylimidazolium species with alcohol 61 (CombiBlocks # JL-5330), in the presence of triethyl amine leads to CICL-250-61. [00433] The numbering paradigm utilized for cyclic-amino-alcohol head groups is based on an ionizable cationic lipid described above that was generated from an acylimidazolide, in this case 6, when it was reacted with 2-dimethylaminoethanol, with the number of the head group- forming alcohol appended, in this case 61. Acylimidazolide 6 was converted to lipid CICL-250 in Example 15. [00434] The utilization of the chemistry described in this Example 20 to generate the active imidazolium species, enables the synthesis of a selection of lipids containing cyclic head groups by substitution of the alcohol 61 (vide supra) with the alcohols appearing in Table 3. Table 3: CICL-250-61 and Substitution of Alcohol 61 with Alcohols 62-77 to Prepare Lipids CICL-25-926 to CICL-250-77 Example 22: Synthesis of 2-(2-(tert-butoxy)-2-oxoethyl)propane-1,3-diyl dinonanoate 78 [00435] To a solution of tert-butyl 4-hydroxy-3-(hydroxymethyl)butanoate (Org. Proc. Res. Dev. 2011, 15, 515; 44.0g, 0.231mol), in acetonitrile (900mL), cooled in an ice water bath under nitrogen, was added nonanoic acid (76.86g, 0.486mol), followed by the addition of DMAP (28.22g, 0.231mol) and EDCl (97.8g, 0.513mol). The mixture was stirred for 1 hour, then was allowed to warm to room temperature and was stirred for 12 hours. The solution was cast into n-heptane (1.40L) and water (0.9L) and the organic phase was separated. The organic phase was washed twice with MeOH:10% aq. citric acid (0.90L), followed by washing twice with a mixture of MeOH:water:triethyl amine (0.90L, 3:1:0.1). The organic phase was then washed with 10% aq. NaCl, dried over Na2SO4, filtered, and concentrated in vacuo to provide 2-(2-(tert-butoxy)-2-oxoethyl)propane-1,3-diyl dinonanoate 78 (90.20g, 96.3% purity by HPLC, 0.185mol, 80% yield) as a pale-yellow viscous liquid. [00436] 1H-NMR (300MHz, CDCl3): δ = 4.12 (m, 4H), 2.53 (m, 1H), 2.29-2.34 (6H), 1.52- 1.64 (4H), 1.46 (s, 9H), 1.16-1.37 (24H), 0.89 (t, J = 7.0Hz, 6H); LCMS: RT = 1.748, calcd. for C27H50O6 minus t-butyl + H+: 415.31. Found: 415.20. Example 23: Synthesis of 4-(nonanoyloxy)-3-((nonanoyloxy)methyl)butanoic acid 79 [00437] To a solution of 78 (90.0g, 96.3% purity, 0.184mol) in toluene (0.41L), cooled in an ice-water bath under nitrogen, was added TFA (208.46g, 1.83mol, 140mL) over a period of 30 minutes. After the addition was complete the mixture was warmed to 15°C and the mixture was stirred for 18 hours. The chilled solution was cast into n-heptane (1.50L) and the resulting solution was extracted with 5% aq. potassium phosphate (2.0L) and the aqueous phase was collected. The organic phase was extracted with MeOH:water:triethyl amine (2.0L, 5:1:0.1), and the combined aq. phases were cast into n-heptane (1.80L) and 1.2M aq. HCl (1.0L). The organic layer was separated, washed with MeOH:water (1.0L, 1:1), dried over Na2SO4, filtered and concentrated in vacuo to afford acid 79 (69.0g, 96.1% purity by HPLC, 0.177mol, 96% yield) as a pale yellow, viscous oil. [00438] 1H-NMR (300MHz, CDCl3): δ = 4.13 (m, 4H), 2.58 (m, 1H), 2.48 (m, 2H), 2.32 (m, 4H), 1.63 (m, 4H), 1.20-1.37 (24H), 0.89 (t, J = 7.0Hz, 6H); LCMS: RT = 1.723, Calcd. for C23H42O6+H+ 415.31. Found 415.30.
Example 24: Synthesis of O'1,O1-((((2-(dimethylamino)ethoxy)carbonyl)azanediyl) bis(ethane-2,1-diyl)) 5,5'-dioctyl bis(3-((nonanoyloxy)methyl)pentanedioate): CICL-309 [00439] The coupling of alcohol 80 (J. Proteome Res.2017, 16, 2457) with nonanoic acid, utilizing EDCl and DMAP in CH3CN, results in triester 81. Deprotection of the t-butyl esters with, TFA in CH2Cl2, provides diacid 82, which is coupled with 1-octanol (1.0 eq, EDCl, DMAP, CH3CN) to give diester-acid 83 after purification. The coupling of acid 83 with diol 3, using EDCl and DMAP in acetonitrile affords 84. Deprotection of the BOC-blocked amine with TFA in CH2Cl2 provides ammonium trifluoracetate salt 85. Treatment of 85 with carbonyl- diimidazole and Et3N in CH2Cl2, results in acyl-imidazole 86. Alkylation of 86 with MeOTf in CH3CN, followed by the reaction of the derived acyl-imidazolium salt with 2- dimethylaminoethanol and (CH3)3N results in CICL-309.
Example 25: Synthesis of 1-(2-(((2-(dimethylamino)ethoxy)carbonyl)(2-((3- ((nonanoyloxy)methyl)-5-(octyloxy)-5-oxopentanoyl)oxy)ethyl)amino)ethyl) 5-octyl 3- (2-(octyloxy)-2-oxoethyl)pentanedioate: CICL-310 [00440] The coupling of acid 83 (1 equiv.) with diol 3 affords mono-esterified alcohol 87 after purification to remove unreacted 3 and bis-esterified 84. Alcohol 87 is coupled with acid 2 utilizing EDCl and DMAP in CH3CN, which results in 88. Deprotection of the BOC-blocked amine with TFA in CH2Cl2 provides ammonium trifluoracetate salt 89. Treatment of 89 with carbonyl-diimidazole and Et3N in CH2Cl2, results in acyl-imidazole 90. Alkylation of 90 with MeOTf in CH3CN, followed by the reaction of the derived acyl-imidazolium salt with 2- dimethylaminoethanol and (CH3)3N results in CICL-310. Example 26: Synthesis of 1-(2-(((2-(dimethylamino)ethoxy)carbonyl)(2-((4- (nonanoyloxy)-3-((nonanoyloxy)methyl)butanoyl)oxy)ethyl)amino)ethyl) 5-octyl 3- ((nonanoyloxy) methyl)pentanedioate: CICL-311 93 CICL-311 [00441] Alcohol 87 is coupled with acid 79 utilizing EDCl and DMAP in CH3CN, which results in 91. Deprotection of the BOC-blocked amine with TFA in CH2Cl2 provides ammonium trifluoracetate salt 92. Treatment of 92 with carbonyl-diimidazole and Et3N in CH2Cl2, results in acyl-imidazole 93. Alkylation of 93 with MeOTf in CH3CN, followed by the reaction of the derived acyl-imidazolium salt with 2-dimethylaminoethanol and (CH3)3N results in CICL-311. [00442] In a similar fashion, utilizing the chemistry of this Example 20, the various acylimidazolides: 10, 43 and 46, as well as acids 13, 37, 48, and 60 are reacted with alcohols 61-77 to create the congeners having the various cyclic head groups on each of the cores described above. Given a desired range of measured pKa of from 6-7, a restriction of the range of calculated pKa (c-pKa) is likely needed to achieve that target measured pKa range. With a measured pKa in formulation (see Table 5) reported for CICI-250 of 6.54 (c-pKa = 8.47) it is clear that a subset of the alcohols utilized in Table 3 will be appropriate partners for coupling to acylimidazolides 10, 43 and 46, as well as acids 13, 37, 48, and 60. The alcohols which are appended to acylimidazolide 6 to provide lipids CICL-250-61 to CICL-250-77, which result in a calculated pKa (c-pKa) of ca. 8 to 9 (Table 3), are utilized to form lipids with the acylimidazolides, and acids cited above. The alcohols thus selected are: 61, 62, 63, 65, 67, 68, 72, and 75. The numbering convention applied to the structures in Table 4, is as described above, is taken from the initial lipid structure created from the listed additional core acylimidazolides 10, 43 and 46, now combined with the selected alcohols listed above, leading to CICL-291-61, when 10 might be combined with alcohol 61, CICL-297-61 for the combination of 43 with alcohol 61, and CICL-298-61 for the combination of 46 with alcohol 61. Table 4. Lipids formed from the Combination of Acylimidazolides 10, 43, 46, and acids 13, 37, 48, 60 with the selected Alcohols 61, 62, 63, 65, 67, 68, 72, 75. [00443] The analysis presented in the preceding paragraph and discussion of adjusting basicity above, which utilized calculated pKa (c-pKa) for cyclic head groups 61-77 attached to the CICL-250 framework (Table 3), which led to a selection of amino-alcohols 61, 62, 63, 65, 67, 68, 72, and 75 as appropriate entities to result in a modification of the measured pKa toward the mid-point of the target pKa range of 6 to 7, resulted in the combination of these amino alcohols with the structural frameworks of CICL-291, CICL-292, CICL-296, CICL-297, CICL-298, CICL-299, and CICL-303 (Table 4) in anticipation of alteration of measured pKa in formulation toward the midpoint of the pKa 6-7 range for these structures. An identical analytical paradigm, starting with calculated pKa, can be performed with the head groups presented in Table 3 to select candidates appropriate for measured pKa alteration for the frameworks of CICL-250, CICL-291, CICL-292, CICL-296, CICL-297, CICL-298, CICL-299, CICL-303, CICL-309, CICL-310, and CICL-311. Example 27. In Vivo Delivery of mRNA by LNP Incorporating CICL-250 as the Ionizable Cationic Lipid [00444] To assess the ability of an ionizable cationic lipids to facilitate in vivo transfection of T cells with mRNA, tLNP incorporating CICL-250, or for comparison, CICL-1, and conjugated to an anti-mouse CD5 antibody were prepared and administered to C57BL/6 mice. The tLNP comprised either CICL-250 or CICL-1, DSPC, cholesterol, DSG-PEG(2000), and DSPE-PEG(2000)-maleimide in the proportions indicated in Table 5 (below) and an N/P ratio of 6. CICL-1 has the structure: [00445] Briefly, to prepare the tLNP, N1-methylpseudouridine (m1ψ)-substituted mCherry mRNA (SEQ ID NO: 2) was encapsulated in LNP using a self-assembly process in which an aqueous solution of mRNA at pH 3.5 was rapidly mixed with a solution of lipids dissolved in ethanol, then followed by stepwise phosphate and Tris buffer dilution and tangential flow filtration (TFF) purification. Then an anti-CD5 mAb was conjugated to the above LNP to generate tLNP. Purified rat anti-mouse CD5 antibody, clone 53-7.3 (BioLegend), was coupled to LNP via N-succinimidyl S-acetylthioacetate (SATA)–maleimide conjugation chemistry. Briefly, LNPs with DSPE-PEG(2000)-maleimide incorporated were formulated and stored at 4°C on the day of conjugation. The antibody was modified with SATA (Sigma-Aldrich) to introduce sulfhydryl groups at accessible lysine residues allowing conjugation to maleimide. SATA was deprotected using 0.5 M hydroxylamine followed by removal of the unreacted components by G-25 Sephadex Quick Spin Protein columns (Roche Applied Science, Indianapolis, IN). The reactive sulfhydryl group on the antibody was then conjugated to maleimide moieties on the LNPs using thioether conjugation chemistry. Purification was performed using Sepharose CL-4B gel filtration columns (Sigma-Aldrich). tLNPs (LNPs conjugated with a targeting antibody) were frozen at -80°C. [00446] The particle size (hydrodynamic diameter) and polydispersity index of the targeted lipid nanoparticles were determined using dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Size measurement was carried out in pH 7.4 Tris buffer at 25°C in disposable capillary cells. A non-invasive back scatter system (NIBS) with a scattering angle of 173° was used for size measurements. mRNA content was determined using a Quant-iTTM RiboGreen RNA assay kit (InvitrogenTM). Encapsulation efficiency was calculated by determining the unencapsulated mRNA content by measuring the fluorescence intensity (Fi) upon the addition of RiboGreen® reagent to the LNP and comparing this value to the total fluorescence intensity (Ft) of the RNA content that is obtained upon lysis of the LNPs by 1% Triton X-100, where % encapsulation = (Ft – Fi)/Ft × 100). After conjugation, tLNP antibody to mRNA weight ratio (binder density) was determined with the BCA (bicinchoninic acid) total protein assay and Ribogreen® assay of mRNA content. Table 5. Physicochemical properties of the tLNP [00447] As seen in Table 5, all of these tLNP compositions had hydrodynamic diameters and polydispersity indices within the acceptable ranges of 50-150 nm and ≤0.2 for PDI. Encapsulation efficiency is acceptable at ≥80% although ≥85% and ≥90% are preferred. Binder density (Ab:mRNA ratio (wt:wt)) is acceptable at ratios of 0.3 to 1.0. [00448] The apparent or measured pKa of ionizable lipid in the lipid nanoparticle was determined using 6-(p-toluidino)-2-naphthalenesulfonic acid sodium salt (TNS salt, Toronto Research Chemicals, Toronto, ON, Canada). Lipid nanoparticles were diluted in 1xDulbecco's PBS to a concentration of 1 mM total lipids. TNS salt was prepared as a 1 mg/mL stock solution in DMSO and then further diluted using distilled water to a working solution of 60 µg/mL (179 mM). Diluted lipid nanoparticle samples were further diluted to 90 µM total lipids in 165 µL of buffered solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, and final TNS concentration of 1.33 µg/mL (4 µM) with the pH ranging from 3.5 to 12.2. Following pipette mixing and incubation at room temperature in the dark for 15 min, fluorescence intensity was measured at room temperature in a BioTek Synergy H1 plate reader using excitation and emission wavelengths of 321 and 445 nm, respectively. The fluorescence signal was blank subtracted and plotted as a function of the pH, then analyzed using a nonlinear (Boltzmann) regression analysis with the apparent pKa determined as the pH giving rise to half maximal fluorescence intensity as calculated by the Henderson−Hasselbalch equation. [00449] The mice were administered tLNPs containing 10 µg of mRNA by tail vein injection and tissue was harvested 24 hours later. As seen in Figure 1A, tLNP comprising DSG- PEG(2000) and either CICL-250 or CICL-1 had similar performance in splenic T cells for both transfection rate and expression level (measured as molecular equivalent of soluble fluorochrome (MESF)). The tLNP incorporating CICL-250 and CICL-1 had similarly low transfection rates (<4%) for CD45- liver cells (hepatocytes) (Figure 1B). The tLNP incorporating CICL-250 and CICL-1 had similarly low levels in liver Kupffer cells (CD45+/CD11+ liver cells) (Figure 1C). [00450] Additional aspects of the disclosure are provided by the following enumerated embodiments, which can be combined in any number and in any combination not technically or logically inconsistent. This is not an exhaustive listing of the embodiments disclosed which include similar embodiments directed to difference species and genera than those exemplified her. Furthermore, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of this invention. Other modifications that can be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of this invention can be utilized in accordance with the teachings herein. Accordingly, this invention is not limited to that precisely as shown and enumerated below. Embodiment 1. An ionizable cationic lipid having a structure of formula M5, wherein: each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl, A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, A5 is absent, O, S, NH, or NCH3 if A4 is C=O, or A5 is C=O if A4 is not C=O, A6 is O, S, NH, NCH3 or (CH2)0-2, A7 is (CH2)0-6, wherein if A6 is O, S, NH, NCH3, A7 is (CH2)2-4, each W is independently CH or N; each R2 is independently O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of a W is O, that W is CH; and wherein when both R3 groups at a beta position of a W are C=O, that W is CH or N; wherein A6 and A7 are not both (CH2)0 unless A5 is C=O, or unless A4 is C=O and A5 is absent; wherein a) A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, or NCH3, A6 is (CH2)1-2, A7 and is (CH2)1-4, or b) A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, or O, A5 is C=O, A6 is O, NH, NCH3 or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, and A7 is (CH2)2-4, or c) A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, NH, or NCH3, A6 is (CH2)1-2, A7 is (CH2)1-4, or 0, wherein the number of contiguous atoms present in a span: O A3 A4 6 5 A A1 A2 X A A7 is in the range from 7-17. Embodiment 2. The ionizable cationic lipid of embodiment 1, wherein in each tail group one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O, and its adjoining R3 is O. Embodiment 3. The ionizable cationic lipid of embodiment 1, wherein in one tail group, one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O and its adjoining R3 is O, and in the other tail group, both R2 groups are O and both R3 groups are C=O. Embodiment 4. The ionizable cationic lipid of embodiment 1, wherein in one tail group, one R2 is O and its adjoining R3 is C=O and the other R2 is C=O and its adjoining R3 is O, and in the other tail group both R2 groups are C=O and both R3 groups are O. Embodiment 5. The ionizable cationic lipid of embodiment 1, wherein in one tail group, both R2 groups are O and both R3 groups are C=O, and in the other tail group, both R2 groups are C=O and both R3 groups are O. Embodiment 6. The ionizable cationic lipid of embodiment 1, wherein in each tail group, both R2 groups are O and both R3 groups are C=O. Embodiment 7. The ionizable cationic lipid of any one of embodiments 1-6, wherein each W is CH. Embodiment 8. The ionizable cationic lipid of embodiment 6, wherein each W is N. Embodiment 9. The ionizable cationic lipid of any one of embodiments 1, 3, 5, or 6 wherein both R3 groups at a beta position of a W are C=O and that W is N. Embodiment 10. The ionizable cationic lipid of any one of embodiments 1, 3, 5, or 6 wherein both R3 groups at a beta position of a W are C=O and that W is CH. Embodiment 11. The ionizable cationic lipid of any one of embodiments 1-5, wherein at least one R3 group at a beta position of a W is O and that W is CH. Embodiment 12. The ionizable cationic lipid of any one of embodiments 1, 4, or 5, wherein both R3 groups at a beta position of a W are O and that W is CH. Embodiment 13. The ionizable cationic lipid of any one of embodiments 1-12, wherein A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, or NCH3, A6 is CH2, A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. Embodiment 14. The ionizable cationic lipid of embodiment 13, wherein A5 is O. Embodiment 15. The ionizable cationic lipid of any one of embodiments 1-12, wherein A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, O, A5 is C=O, A6 is O, NH, NCH3, or CH2, A7 is (CH) 2-6. Embodiment 16. The ionizable cationic lipid of embodiment 15, wherein A4 is NH. Embodiment 17. The ionizable cationic lipid of embodiment 15, wherein A4 is NH and A6 is O. Embodiment 18. The ionizable cationic lipid of embodiment 15, wherein A4 is NH and A6 is CH2. Embodiment 19. The ionizable cationic lipid of embodiment 15, wherein A4 is CH2. Embodiment 20. The ionizable cationic lipid of embodiment 15, wherein A4 is CH2 and A6 is O. Embodiment 21. The ionizable cationic lipid of embodiment 15, wherein A4 is O. Embodiment 22. The ionizable cationic lipid of embodiment 15, wherein A4 is O and A6 is CH2. Embodiment 23. The ionizable cationic lipid of embodiment 15, wherein A4 is NCH3. Embodiment 24. The ionizable cationic lipid of embodiment 15, wherein A4 is NCH3 and A6 is CH2. Embodiment 25. The ionizable cationic lipid of any one of embodiments 1-12, wherein A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, NH, NCH3, A6 is (CH2)1-2, A7 is (CH2)1- 4. Embodiment 26. The ionizable cationic lipid of embodiment 25, wherein A5 is O. Embodiment 27. The ionizable cationic lipid of any one of embodiments 1-25, wherein O the number of contiguous atoms present in at least one span: is in the range of 10 to 17. Embodiment 28. The ionizable cationic lipid of any one of embodiments 1-12, wherein A1 absent, A6 is (CH2)0, A7 is (CH2)0, and Y is Embodiment 29. The ionizable cationic lipid of embodiment 28, wherein the number of O contiguous atoms present in at least one span: is in the range of 7- 10. Embodiment 30. The ionizable cationic lipid of any one of embodiments 1-12, wherein A1 Embodiment 31. The ionizable cationic lipid of embodiment 30, wherein A4 is NH. Embodiment 32. The ionizable cationic lipid of embodiment 30, wherein A4 is CH2. Embodiment 33. The ionizable cationic lipid of embodiment 30, wherein A4 is O. Embodiment 34. The ionizable cationic lipid of embodiment 30, wherein A4 is NCH3. Embodiment 35. The ionizable cationic lipid of any one of embodiments 1-12, wherein A1 absent, A6 is (CH2)0, A7 is (CH2)0, and Y Embodiment 36. The ionizable cationic lipid of any one of embodiments 30-35, wherein O 3 4 6 the number of contiguous atoms present in at least one span: A1 A A A2 X A5 A A7 is in the range of 7-11. Embodiment 37. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 39. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Z N Y is and Z is a bond. Embodiment 40. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond. Embodiment 41. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein OCH3 Z N Y is and Z is a bond. Embodiment 42. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond. Embodiment 43. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 44. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 46. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond. Embodiment 47. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond. Embodiment 48. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond. Embodiment 49. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 51. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond. Embodiment 52. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 54. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 56. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 58. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 60. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein Embodiment 62. The ionizable cationic lipid of any one of embodiments 1 to 36, wherein bond. Embodiment 63. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is independently C7-C10 alkyl or C7-C9 alkyl. Embodiment 64. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is independently a linear C7-C11 alkyl, e.g., a linear C7-C10 alkyl, or a linear C7-C9 alkyl. Embodiment 65. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is independently (CH2)6-8CH3. Embodiment 66. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is (CH2)7CH3. Embodiment 67. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is independently a linear C7-C11 alkenyl, e.g., a linear C7-C10 alkenyl, or a linear C7-C9 alkenyl. Embodiment 68. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is a linear C8 alkenyl. Embodiment 69. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is independently a branched C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. Embodiment 70. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is a branched C8 alkyl. Embodiment 71. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is independently a branched C7-C11 alkenyl, e.g., C7-C10 alkenyl, or C7-C9 alkenyl. Embodiment 72. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein each R1 is a branched C8 alkenyl. Embodiment 73. The ionizable cationic lipid of any one of embodiments 1 to 62, wherein R1 is a branched alkyl or alkenyl, the branch point is positioned so that ester carbonyls are not in an α position relative to the branch point, for example they are in a β position relative to the branch point. Embodiment 74. The ionizable cationic lipid of any one of embodiments 1 to 73, wherein each R1 is the same. Embodiment 75. An ionizable cationic lipid having a structure of formula M4, wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl, A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, A5 is absent, O, S, NH, NCH3, or C=O, A6 is O, S, NH, NCH3 or (CH2)0-2, A7 is (CH2)0-6, wherein if A6 is O, S, NH, NCH3, A7 is (CH2)2-4, W is CH or N, wherein A6 and A7 are not both (CH2)0 unless A5 is C=O, or unless A4 is C=O and A5 is absent; wherein a) A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, or NCH3, A6 is (CH2)1-2, and A7 is (CH2)1-4, or b) A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, or O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4, or c) A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, NH, or NCH3, A6 is (CH2)1-2, and A7 is (CH2)1-4, or 0, absent, A6 is (CH2)0, A7 is wherein the number of contiguous atoms present in a span: the range from 7-17. Embodiment 76. The ionizable cationic lipid of embodiment 75, wherein A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, NCH3, A6 is (CH2)1-2, A7 is (CH2)1-4. Embodiment 77. The ionizable cationic lipid of embodiment 76, wherein A5 is O. Embodiment 78. The ionizable cationic lipid of embodiment 75, wherein A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, or O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH2)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. Embodiment 79. The ionizable cationic lipid of embodiment 78, wherein A4 is NH. Embodiment 80. The ionizable cationic lipid of embodiment 78, wherein A4 is NH and A6 is O. Embodiment 81. The ionizable cationic lipid of embodiment 78, wherein A4 is NH and A6 is CH2. Embodiment 82. The ionizable cationic lipid of embodiment 78, wherein A4 is CH2. Embodiment 83. The ionizable cationic lipid of embodiment 78, wherein A4 is CH2 and A6 is O. Embodiment 84. The ionizable cationic lipid of embodiment 78, wherein A4 is O. Embodiment 85. The ionizable cationic lipid of embodiment 78, wherein A4 is O and A6 is CH2. Embodiment 86. The ionizable cationic lipid of embodiment 78, wherein A4 is NCH3. Embodiment 87. The ionizable cationic lipid of embodiment 78, wherein A4 is NCH3 and A6 is CH2. Embodiment 88. The ionizable cationic lipid of embodiment 75, wherein A1 is (CH2)2, A3 is (CH2)1-4, X is C-CH3, A4 is C=O, A5 is O, NH, NCH3, A6 is (CH2)1-2, A7 is (CH2)1-4. Embodiment 89. The ionizable cationic lipid of embodiment 88, wherein A5 is O. Embodiment 90. The ionizable cationic lipid of any one of embodiments 75-89, wherein O A3 4 6 the number of contiguous atoms present in at least one span: A1 A2 A X A5 A A7 is in the range of 10 to 17. Embodiment 91. The ionizable cationic lipid of embodiment 75, wherein A1 is CH2, A3 is C=O, A5 is absent, A6 is (CH2)0, A7 is (CH2)0, and Y is Embodiment 92. The ionizable cationic lipid of embodiment 91, wherein the number of O A3 A4 6 contiguous atoms present in at least one span: A1 A2 X A5 A A7 is in the range of 7- 10. Embodiment 93. The ionizable cationic lipid of embodiment 75, wherein A1 is CH2, A3 is and Y is Embodiment 94. The ionizable cationic lipid of embodiment 93, wherein A4 is NH. Embodiment 95. The ionizable cationic lipid of embodiment 93, wherein A4 is CH2. Embodiment 96. The ionizable cationic lipid of embodiment 93, wherein A4 is O. Embodiment 97. The ionizable cationic lipid of embodiment 93, wherein A4 is NCH3. Embodiment 98. The ionizable cationic lipid of embodiment 75, wherein A1 is (CH2)2, A3 absent, A6 is (CH2)0, A7 is (CH2)0, and Y is Embodiment 99. The ionizable cationic lipid of any one of embodiments 91-98, wherein O A3 A4 A6 the number of contiguous atoms present in at least one span: A1 A2 X A5 A7 is in the range of 7-11. Embodiment 100. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond. Embodiment 101. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein (CH2)0-1 CH 3 Z N Y is (CH2)0-1 CH 3 and Z is a bond. Embodiment 102. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond. Embodiment 103. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond. Embodiment 104. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein OCH3 Z N Y is and Z is a bond. Embodiment 105. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 107. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 109. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 110. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond. Embodiment 111. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond. Embodiment 112. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 114. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 116. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 118. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond. Embodiment 119. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 121. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 123. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein Embodiment 125. The ionizable cationic lipid of any one of embodiments 75 to 99, wherein bond. Embodiment 126. The ionizable cationic lipid of any one of embodiments 75 to 125, wherein W is CH. Embodiment 127. The ionizable cationic lipid of any one of embodiments 75 to 125, wherein W is N. Embodiment 128. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is independently C7-C10 alkyl or C7-C9 alkyl. Embodiment 129. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is independently a linear C7-C11 alkyl, e.g., a linear C7-C10 alkyl, or a linear C7-C9 alkyl. Embodiment 130. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is independently (CH2)6-8CH3. Embodiment 131. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein R1 is (CH2)7CH3. Embodiment 132. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is independently a linear C7-C11 alkenyl, e.g., a linear C7-C10 alkenyl, or a linear C7-C9 alkenyl. Embodiment 133. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is a linear C8 alkenyl. Embodiment 134. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is independently a branched C7-C11 alkyl, e.g., C7-C10 alkyl, or C7-C9 alkyl. Embodiment 135. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is a branched C8 alkyl. Embodiment 136. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is independently a branched C7-C11 alkenyl, e.g., C7-C10 alkenyl, or C7-C9 alkenyl. Embodiment 137. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein each R1 is a branched C8 alkenyl. Embodiment 138. The ionizable cationic lipid of any one of embodiments 75 to 127, wherein R1 is a branched alkyl or alkenyl, the branch point is positioned so that ester carbonyls are not in an α position relative to the branch point, for example they are in a β position relative to the branch point. Embodiment 139. The ionizable cationic lipid of any one of embodiments 75 to 138, wherein each R1 is the same. Embodiment 140. The ionizable cationic lipid of embodiment 75 having the structure CICL-250: CICL-250 . Embodiment 141. The ionizable cationic lipid of embodiment 75 having the structure CICL-291: . Embodiment 142. The ionizable cationic lipid of embodiment 75 having the structure CICL-292: . Embodiment 143. The ionizable cationic lipid of embodiment 75 having the structure CICL-293: . Embodiment 144. The ionizable cationic lipid of embodiment 75 having the structure CICL-294: Embodiment 145. having the structure CICL-295: . Embodiment 146. having the structure CICL-296: . Embodiment 147. The ionizable cationic lipid of embodiment 75 having the structure CICL-297: . Embodiment 148. The ionizable cationic lipid of embodiment 75 having the structure CICL-298: . Embodiment 149. The ionizable cationic lipid of embodiment 75 having the structure CICL-299: . Embodiment 150. The ionizable cationic lipid of embodiment 75 having the structure CICL-300: . Embodiment 151. The ionizable cationic lipid of embodiment 75 having the structure CICL-301: . Embodiment 152. The ionizable cationic lipid of embodiment 75 having the structure CICL-302: . Embodiment 153. The ionizable cationic lipid of embodiment 75 having the structure CICL-303: . Embodiment 154. The ionizable cationic lipid of embodiment 1 having a structure as shown in Table 2. Embodiment 155. The ionizable cationic lipid of embodiment 1 having a structure as shown in Table 3. Embodiment 156. The ionizable cationic lipid of embodiment 1 having a structure as shown in Table 4. Embodiment 157. The ionizable cationic lipid of any one of embodiments 1 to 156 having a -pKa (calculated pKa) in the range of from about 6, 7, or 8 to about 9, 10, or 11. Embodiment 158. The ionizable cationic lipid of any one of embodiments 1 to 156 having a c-pKa ranging from about 6 to about 10, about 7 to about 10, about 8 to about 10, about 8 to about 9, 6 to 10, 7 to 10, 8 to 10, or 8 to 9. Embodiment 159. The ionizable cationic lipid of any one of embodiments 1 to 156 having a c-pKa ranging from about 8.2 to about 9.0 or from 8.2 to 9.0. Embodiment 160. The ionizable cationic lipid of any one of embodiments 1 to 156 having a c-pKa ranging from about 8.4 to about 8.7 or from 8.4 to 8.7. Embodiment 161. The ionizable cationic lipid of any one of embodiments 1 to 160 having a cLogD ranging from about 9 to about 18, for example, ranging from about 10 to about 18, or about 10 to about 16, to about 10 to about 14, or about 11 to about 18, or about 11 to about 15, or about 11 to about 14, or about 12 to about 14. Embodiment 162. The ionizable cationic lipid of any one of embodiments 1 to 160 having a cLogD ranging from 9 to 18, for example, ranging from 10 to 18, or 10 to 16, to 10 to 14, or 11 to 18, or 11 to 15, or 11 to 14, or 12 to 14. Embodiment 163. The ionizable cationic lipid of any one of embodiments 1 to 160 having a cLogD is about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18. Embodiment 164. The ionizable cationic lipid of any one of embodiments 1 to 160 having a cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4. Embodiment 165. The ionizable cationic lipid of any one of embodiments 1 to 160 having a c-pKa ranging from about 8 to about 11 or from 8 to 11 and a cLogD ranging from about 9 to about 18 or from 9 to 18. Embodiment 166. The ionizable cationic lipid of any one of embodiments 1 to 160 having a c-pKa ranging from about 8.4 to about 8.7 or from 8.4 to 8.7 and cLogD ranging from about 13.6 to about 14.4 or from 13.6 to 14.4. Embodiment 167. A lipid nanoparticle (LNP), comprising at least one ionizable cationic lipid of any one of embodiments 1-166. Embodiment 168. A targeted lipid nanoparticle (tLNP), comprising a lipid nanoparticle of embodiment 167 and a functionalized PEG-lipid, wherein the functionalized PEG-lipid has been conjugated with a binding moiety. Embodiment 169. The LNP of embodiment 167 or the tLNP of embodiment 168, further comprising one or more of a phospholipid, a sterol, a co-lipid, an unfunctionalized and/or a functionalized PEG-lipid, or combinations thereof. Embodiment 170. The LNP or tLNP of embodiment 169, wherein the phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof. Embodiment 171. The LNP or tLNP of embodiment 169 or 170 wherein the sterol comprises cholesterol, campesterol, sitosterol, stigmasterol, or combinations thereof. Embodiment 172. The LNP or tLNP of any one of embodiments 169-170, wherein the co- lipid comprises cholesterol hemisuccinate (CHEMS) or a quaternary ammonium head group containing lipid. Embodiment 173. The LNP or tLNP of embodiment 172, wherein the quaternary ammonium head group containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3β-(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof. Embodiment 174. The LNP of any one of embodiments 169-173 or the tLNP of any one of embodiments 168-173, wherein the unfunctionalized PEG-lipid and/or the functionalized PEG-lipid comprises a PEG moiety of 1000-5000 Da molecular weight (MW). Embodiment 175. The LNP of any one of embodiments 169-174 or the tLNP of any one of embodiments 168-174, wherein the unfunctionalized PEG-lipid and/or the functionalized PEG-lipid comprises fatty acids with a fatty acid chain length of C14-C18. Embodiment 176. The LNP of any one of embodiments 169-175 or the tLNP of any one of embodiments 168-175, wherein the unfunctionalized PEG-lipid and/or the functionalized PEG-lipid comprises DMG-PEG2000 (1,2-dimyristoyl-rglycero-3-methoxypolyethylene glycol- 2000), DPG-PEG2000 (1,2-dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG- PEG2000 (1,2-distearoyl-glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2- dioleoyl-glycero-3-methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl- glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE- PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol- 2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), or combinations thereof. Embodiment 177. The LNP or tLNP of any one of embodiments 169-176, comprising a phospholipid in an amount in the range from 7 to 30 mol%. Embodiment 178. The LNP or tLNP of any one of embodiments 169-177, comprising a sterol in an amount in the range from 20 to 45 mol%. Embodiment 179. The LNP or tLNP of any one of embodiments 169-178, comprising at least one co-lipid in an amount in the range from 1 to 30 mol%. Embodiment 180. The LNP or tLNP of any one of embodiments 169-179, comprising at least one unfunctionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%. Embodiment 181. The LNP or tLNP of any one of embodiments 169-180, comprising at least one functionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%. Embodiment 182. The tLNP of any one of embodiments 169-181, wherein the binding moiety comprises an antigen, a ligand-binding domain of a receptor, a receptor ligand, an antibody, or an antigen binding domain of an antibody. Embodiment 183. The LNP of any one of embodiments 167 or 169-182 or the tLNP of any one of embodiments 168-182, further comprising a nucleic acid molecule payload. Embodiment 184. The LNP or tLNP of embodiment 183, wherein the weight ratio of total lipid to nucleic acid is 10:1 to 50:1. Embodiment 185. The LNP or tLNP of embodiment 183, wherein the N/P ratio is from 3 to 9. Embodiment 186. The LNP or tLNP of any one of embodiments 183-185, wherein the nucleic acid molecule is an mRNA. Embodiment 187. The tLNP of any one of embodiments 182-186, wherein the binding moiety is a whole antibody and the ratio of antibody to nucleic acid molecule is from about 0.3 to about 1.0 (w/w). Embodiment 188. The tLNP of any one of embodiments 168-187, wherein the binding moiety is a F(ab’) or F(ab’) analog. Embodiment 189. The tLNP of any one of embodiments 182-188, wherein the tLNP is targeted to a T cell. Embodiment 190. The tLNP of any one of embodiments 182-189, wherein the tLNP is targeted to a CD8+ T cell. Embodiment 191. The tLNP of any one of embodiments 182-188, wherein the tLNP is targeted to an HSC. Embodiment 192. The tLNP of any one of embodiments 182-188, wherein the tLNP is targeted to a CD117+ cell. Embodiment 193. A method of delivering a biologically active payload (e.g., one or more species of nucleic acid molecule) into a cell comprising contacting the cell with the LNP or tLNP any one of embodiments 183-192. Embodiment 194. A lipid having the structure of formula I5-1, wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; each R2 is independent O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=O, W is CH or N; A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, and R4 is H or a protecting group. Embodiment 195. The lipid of embodiment 194, wherein R1, R2, R3, and W are as otherwise described herein. Embodiment 196. The lipid of embodiment 194 or embodiment 195, wherein R4 is H. Embodiment 197. The lipid of embodiment 194 or embodiment 195, wherein R4 is a protecting group (e.g., t-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), or a trimethylsilylethoxycarbonyl moiety. Embodiment 198. The lipid of any one of embodiments 194-197, wherein when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. Embodiment 199. The lipid of any one of embodiments 194-197, wherein when A1 and A3 are CH2, then X is CH, and A4 is NH2. Embodiment 200. The lipid of any one of embodiments 194-197, wherein when A1 is CH2 and A3 is CH2 or CH2CH2, and X is CH, then A4 is NH, NCH3, or O. Embodiment 201. The lipid of any one of embodiments 194-197, wherein when A1 is CH2, A3 is CH2CH2, X is CH, and A4 is O. Embodiment 202. The lipid of any one of embodiments 194-197, wherein when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NH. Embodiment 203. The lipid of any one of embodiments 194-197, wherein when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NCH3. Embodiment 204. A lipid having the structure of formula I5-2, wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; each R2 is independent O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=O, W is CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, and A4 is CH2, NH, NCH3, or O, and Embodiment 205. The lipid of embodiment 204, wherein R1, R2, R3, and W are as otherwise described herein. Embodiment 206. The lipid of embodiment 204 or embodiment 205, wherein R5 is OH. Embodiment 207. The lipid of embodiment 204 or embodiment 205, wherein R5 is . Embodiment 208. The lipid of any one of embodiments 204-207, wherein when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. Embodiment 209. The lipid of any one of embodiments 204-207, wherein A1 and A3 are Embodiment 210. The lipid of any one of embodiments 204-207, wherein A1 and A3 are CH2, X is CH, A5 is CH2, and R5 is OH. Embodiment 211. A lipid having the structure of formula I5-3, wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; each R2 is independent O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of W is O, W is CH; and wherein when both R3 groups at a beta position of W are C=O, W is CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, and X is N, CH, or C-CH3; and Embodiment 212. The lipid of embodiment 211, wherein R1, R2, R3, and W are as otherwise described herein. Embodiment 213. The lipid of embodiment 211 or embodiment 212, wherein R6 is OH. Embodiment 214. The lipid of embodiment 211 or embodiment 212, wherein R6 is . Embodiment 215. The lipid of any one of embodiments 211-214, wherein A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is C-CH3. Embodiment 216. The lipid of any one of embodiments 211-214, wherein A1 is CH2, A3 is CH2CH2, X is C-CH3, and R6 is OH. Embodiment 217. The lipid of any one of embodiments 211-214, wherein A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is N. Embodiment 218. The lipid of any one of embodiments 211-214, wherein A1 is CH2, A4 is Embodiment 219. A lipid having the structure of formula I4-1, wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, and R4 is H or a protecting group. Embodiment 220. The lipid of embodiment 219, wherein R1 and W are as otherwise described herein. Embodiment 221. The lipid of embodiment 219 or embodiment 220, wherein R4 is H. Embodiment 222. The lipid of embodiment 219 or embodiment 220, wherein R4 is a protecting group (e.g., t-butoxycarbonyl (BOC), benzyloxycarbonyl (Cbz), or a trimethylsilylethoxycarbonyl moiety. Embodiment 223. The lipid of any one of embodiments 219-222, wherein when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. Embodiment 224. The lipid of any one of embodiments 219-222, wherein when A1 and A3 are CH2, then X is CH, and A4 is NH2. Embodiment 225. The lipid of any one of embodiments 219-222, wherein when A1 is CH2 and A3 is CH2 or CH2CH2, and X is CH, then A4 is NH, NCH3, or O. Embodiment 226. The lipid of any one of embodiments 219-222, wherein when A1 is CH2, A3 is CH2CH2, X is CH, and A4 is O. Embodiment 227. The lipid of any one of embodiments 219-222, wherein when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NH. Embodiment 228. The lipid of any one of embodiments 219-222, wherein when A1 is CH2, A3 is CH2CH2, X is C-CH3, and A4 is NCH3. Embodiment 229. A lipid having the structure of formula I4-2, wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, and A4 is CH2, NH, NCH3, or O, and Embodiment 230. The lipid of embodiment 229, wherein R1 and W are as otherwise described herein. Embodiment 231. The lipid of embodiment 229 or embodiment 230, wherein R5 is OH. Embodiment 232. The lipid of embodiment 229 or embodiment 230, wherein R5 is . Embodiment 233. The lipid of any one of embodiments 229-232, wherein when A1 and A3 are CH2, then X is CH, A4 is CH2, NH, NCH3, or O. Embodiment 234. The lipid of any one of embodiments 229-232, wherein A1 and A3 are Embodiment 235. The lipid of any one of embodiments 229-232, wherein A1 and A3 are CH2, X is CH, A5 is CH2, and R5 is OH. Embodiment 236. A lipid having the structure of formula I4-3, wherein each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl; each W is independently CH or N; A1 is CH2 or CH2CH2, A2 is O, A3 is CH2 or CH2CH2, wherein A3 is not CH2 if X is N, and X is N, CH, or C-CH3; and Embodiment 237. The lipid of embodiment 236, wherein R1 and W are as otherwise described herein. Embodiment 238. The lipid of embodiment 236 or embodiment 237, wherein R6 is OH. Embodiment 239. The lipid of embodiment 236 or embodiment 237, wherein R6 is . Embodiment 240. The lipid of any one of embodiments 236-239, wherein A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is C-CH3. Embodiment 241. The lipid of any one of embodiments 236-239, wherein A1 is CH2, A3 is CH2CH2, X is C-CH3, and R6 is OH. Embodiment 242. The lipid of any one of embodiments 236-239, wherein A1 is CH2 or CH2CH2, A3 is CH2 or CH2CH2, and X is N. Embodiment 243. The lipid of any one of embodiments 236-239, wherein A1 is CH2, A4 is Embodiment 244. A method of treating a disease or disorder comprising administration of a tLNP of any one of embodiments 183-190 to a subject in need thereof. Embodiment 245. The method of embodiment 244, wherein the disease or disorder is an autoimmune disease. Embodiment 246. The method of embodiment 244, wherein the autoimmune disease is a T cell-mediated autoimmunity or a B-cell mediated autoimmunity. Embodiment 247. The method of embodiment 244, wherein the disease or disorder is a rejection of an allogeneic organ or tissue graft. Embodiment 248. The method of embodiment 244, wherein the disease or disorder is cancer. Embodiment 249. The method of embodiment 244, wherein the disease or disorder is a fibrotic disease or disorder. Embodiment 250. The method of embodiment 244, wherein the disease or disorder is a graft versus host disease (GVHD). Embodiment 251. A method of treating a disease or disorder comprising administration of a tLNP of any one of embodiments 183-188 or 191-192 to a subject in need thereof. Embodiment 252. The method of embodiment 251, wherein the disease or disorder is a genetic disease or disorder. Z N [00451] Throughout this disclosure embodiments are described in which Y is , (wherein Z is a bond). There are parallel embodiments to each of these embodiments incorporating each other substituent of Y disclosed herein. Throughout this disclosure embodiments are described in which R1 is linear C8 alkyl. There are parallel embodiments to each of these embodiments incorporating each other substituent of R1 disclosed herein. [00452] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. [00453] While some embodiments have been illustrated and described in detail in the appended drawings and the foregoing description, such illustration and description are to be considered illustrative and not restrictive. Other variations to the disclosed embodiments can be understood and effected in practicing the claims, from a study of the drawings, the disclosure, and the appended claims. The mere fact that certain measures or features are recited in mutually different dependent claims does not indicate that the combination of these measures or features cannot be used. Any reference signs in the claims should not be construed as limiting the scope.
APPENDIX A

Claims

We claim: 1. An ionizable cationic lipid having a structure of formula M5, wherein: each R1 is independently a C7-C11 alkyl or a C7-C11 alkenyl, A1 is (CH2)1-2, A2 is O, A3 is (CH2)1-5, wherein A3 is not CH2 if X is N, X is N, CH, or C-CH3, A4 is CH2, C=O, NH, NCH3, or O, A5 is absent, O, S, NH, or NCH3 if A4 is C=O, or A5 is C=O if A4 is not C=O, A6 is O, S, NH, NCH3 or (CH2)0-2, A7 is (CH2)0-6, wherein if A6 is O, S, NH, NCH3, A7 is (CH2)2-4, each W is independently CH or N; each R2 is independently O or C=O, wherein at least one R2 is O; each R3 is independently O or C=O, wherein when a R2 is O then its adjoining R3 is C=O, or when a R2 is C=O then its adjoining R3 is O; wherein when at least one R3 at a beta position of a W is O, that W is CH; and wherein when both R3 groups at a beta position of a W are C=O, that W is CH or N; wherein A6 and A7 are not both (CH2)0 unless A5 is C=O, or unless A4 is C=O and A5 is absent; wherein a) A1 is CH2, A3 is (CH2)2-5, X is N, A4 is C=O, A5 is O, S, NH, or NCH3, A6 is (CH2)1-2, and A7 is (CH2)1-4, or b) A1 is CH2, A3 is (CH2)1-4, X is CH, A4 is CH2, NH, NCH3, O, A5 is C=O, A6 is O, NH, NCH3, or CH2, and A7 is (CH)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4, or e) A1 is CH2, A3 is (CH2)1-5, X is CH, A4 is CH2, NH, NCH3 or O, A5 is C=O, A6 is 2 0 7 Z N N (CH2)0-3CH3 (CH ) , A is (CH2)0, and Y is , or f) A1 is (CH2)2, A3 is (CH2)1-5, X is CCH3, A4 is C=O, A5 is absent, A6 is (CH2)0, A7 is Z N N (CH2)0-3CH3 (CH2)0, and Y is ; wherein the number of contiguous atoms present in a span: O A3 A4 A6 A1 A2 X A5 A7 is in the range from 7-17. 2. The ionizable cationic lipid of claim 1, wherein in each tail group one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O, and its adjoining R3 is O. 3. The ionizable cationic lipid of claim 1, wherein in one tail group, one R2 is O and its adjoining R3 is C=O, and the other R2 is C=O and its adjoining R3 is O, and in the other tail group, both R2 groups are O and both R3 groups are C=O. 4. The ionizable cationic lipid of claim 1, wherein in one tail group, one R2 is O and its adjoining R3 is C=O and the other R2 is C=O and its adjoining R3 is O, and in the other tail group both R2 groups are C=O and both R3 groups are O. 5. The ionizable cationic lipid of claim 1, wherein in one tail group, both R2 groups are O and both R3 groups are C=O, and in the other tail group, both R2 groups are C=O and both R3 groups are O. 6. The ionizable cationic lipid of claim 1, wherein in both tail groups, both R2 groups are O and both R3 groups are C=O. 7. The ionizable cationic lipid of any one of claims 1-5, wherein at least one R3 at a beta position of a W is O and that W is CH. 8. The ionizable cationic lipid of any one of claims 1, 4, or 5, wherein both R3 groups at a beta position of a W are O and that W is CH. 9. The ionizable cationic lipid of any one of claims 1, 3, 5, or 6, wherein both R3 groups at a beta position of a W are C=O and that W is N. 10. The ionizable cationic lipid of any one of claims 1, 3, 5, or 6, wherein both R3 groups at a beta position of a W are C=O and that W is CH. 11. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is CH2, A3 is (CH2)2- 5, X is N, A4 is C=O, A5 is O, S, NH, NCH3, A6 is (CH2)1-2, A7 is (CH2)1-4. 12. The ionizable cationic lipid of claim 11, wherein A5 is O. 13. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is CH2, A3 is (CH2)1- 4, X is CH, A4 is CH2, NH, NCH3, or O, A5 is C=O, A6 is O, NH, NCH3, or CH2, A7 is (CH)0-6, wherein if A6 is O, NH, NCH3, A7 is (CH2)2-4. 14. The ionizable cationic lipid of claim 13, wherein A4 is NH. 15. The ionizable cationic lipid of claim 13, wherein A4 is NH and A6 is O. 16. The ionizable cationic lipid of claim 13, wherein A4 is NH and A6 is CH2. 17. The ionizable cationic lipid of claim 13, wherein A4 is CH2. 18. The ionizable cationic lipid of claim 13, wherein A4 is CH2 and A6 is O. 19. The ionizable cationic lipid of claim 13, wherein A4 is O. 20. The ionizable cationic lipid of claim 13, wherein A4 is O and A6 is CH2. 21. The ionizable cationic lipid of claim 13, wherein A4 is NCH3. 22. The ionizable cationic lipid of claim 13, wherein A4 is NCH3 and A6 is CH2. 23. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is (CH2)2, A3 is (CH2)1- 4, X is C-CH3, A4 is C=O, A5 is O, NH, NCH3, A6 is (CH2)1-2, A7 is (CH2)1-4. 24. The ionizable cationic lipid of claim 23, wherein A5 is O. 25. The ionizable cationic lipid of any one of claims 7-24, wherein the number of O contiguous atoms present in at least one span: is in the range of 10 to 17. Z N 26. The ionizable cationic lipid of any one of claims 7-25, wherein Y is . 27. The ionizable cationic lipid of any one of claims 7-25, wherein Y is . 28. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is CH2, A3 is (CH2)2- 5, X is N, A4 is C=O, A5 is absent, 29. The ionizable cationic lipid of claim 28, wherein the number of contiguous atoms O 3 4 6 present in at least one span: A1 A2A A A X A5 A7 is in the range of 7-10. 30. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is CH2, A3 is (CH2)1- 5, X is CH, A4 is CH2, NH, NCH3 or O, A5 is C=O, A6 is (CH2)0, A7 is (CH2)0, and Y is . 31. The ionizable cationic lipid of claim 30, wherein A4 is NH. 32. The ionizable cationic lipid of claim 30, wherein A4 is CH2. 33. The ionizable cationic lipid of claim 30, wherein A4 is O. 34. The ionizable cationic lipid of claim 30, wherein A4 is NCH3. 35. The ionizable cationic lipid of any one of claims 7-10, wherein A1 is (CH2)2, A3 is (CH2)1- Z N N (CH2)0-3CH3 5, X is CCH3, A4 is C=O, A5 is absent, A6 is (CH2)0, A7 is (CH2)0, and Y is . 36. The ionizable cationic lipid of any one of claims 30-25, wherein the number of O A3 A4 A6 contiguous atoms present in at least one span: A1 A2 X A5 A7 is in the range of 7- 11. 37. The ionizable cationic lipid of any one of claims 7-36, wherein each R1 is (CH2)7CH3. 38. The ionizable cationic lipid of claim 6 having the structure CICL-250: CICL-250 . 39. The ionizable cationic lipid of claim 6 having the structure CICL-291: . 40. The ionizable cationic lipid of claim 6 having the structure CICL-292: . 41. The ionizable cationic lipid of claim 6 having the structure CICL-293: . 42. The ionizable cationic lipid of claim 6 having the structure CICL-294: 43. The ionizable cationic lipid of claim 6 having the structure CICL-295: . 44. The ionizable cationic lipid of claim 6 having the structure CICL-296: . 45. The ionizable cationic lipid of claim 6 having the structure CICL-297: . 46. The ionizable cationic lipid of claim 6 having the structure CICL-298: . 47. The ionizable cationic lipid of claim 6 having the structure CICL-299: . 48. The ionizable cationic lipid of claim 6 having the structure CICL-300: . 49. The ionizable cationic lipid of claim 6 having the structure CICL-301: CICL-301 . 50. The ionizable cationic lipid of claim 6 having the structure CICL-302: . 51. The ionizable cationic lipid of claim 6 having the structure CICL-303: . 52. A lipid nanoparticle (LNP), comprising at least one ionizable cationic lipid of any one of claims 1-51. 53. A targeted lipid nanoparticle (tLNP), comprising a lipid nanoparticle of claim 52 and a functionalized PEG-lipid, wherein the functionalized PEG-lipid has been conjugated with a binding moiety. 54. The LNP of claim 52 or the tLNP of claim 53, further comprising one or more of a phospholipid, a sterol, a co-lipid, an unfunctionalized and/or a functionalized PEG-lipid, or combinations thereof. 55. The LNP or tLNP of claim 54, wherein the phospholipid comprises dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), or 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC), or a combination thereof. 56. The LNP or tLNP of claim 54 or 55 wherein the sterol comprises cholesterol, campesterol, sitosterol, stigmasterol, or combinations thereof. 57. The LNP or tLNP of any one of claims 54-56, wherein the co-lipid comprises cholesterol hemisuccinate (CHEMS) or a quaternary ammonium head group containing lipid. 58. The LNP or tLNP of claim 57, wherein the quaternary ammonium head group containing lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-(1-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), or 3β-(N-(N',N'- Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), or combinations thereof. 59. The LNP of any one of claims 54-58 or the tLNP of any one of claims 53-58, wherein the unfunctionalized PEG-lipid and/or the functionalized PEG-lipid comprises a PEG moiety of 1000-5000 Da molecular weight (MW). 60. The LNP of any one of claims 54-59 or the tLNP of any one of claims 53-59, wherein the unfunctionalized PEG-lipid and/or the functionalized PEG-lipid comprises fatty acids with a fatty acid chain length of C14-C18. 61. The LNP of any one of claims 54-60 or the tLNP of any one of claims 53-60, wherein the unfunctionalized PEG-lipid and/or the functionalized PEG-lipid comprises DMG-PEG2000 (1,2-dimyristoyl-rglycero-3-methoxypolyethylene glycol-2000), DPG-PEG2000 (1,2- dipalmitoyl-glycero-3-methoxypolyethylene glycol-2000), DSG-PEG2000 (1,2-distearoyl- glycero-3-methoxypolyethylene glycol-2000), DOG-PEG2000 (1,2-dioleoyl-glycero-3- methoxypolyethylene glycol-2000), DMPE-PEG200 (1,2-dimyristoyl-glycero-3- phosphoethanolamine-3-methoxypolyethylene glycol-2000), DPPE-PEG2000 (1,2- dipalmitoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol-2000), DSPE- PEG2000 (1,2-distearoyl-glycero-3-phosphoethanolamine-3-methoxypolyethylene glycol- 2000), DOPE-PEG2000 (1,2-dioleoyl-glycero-3-phosphoethanolamine-3- methoxypolyethylene glycol-2000), or combinations thereof. 62. The LNP or tLNP of any one of claims 54-61, comprising a phospholipid in an amount in the range from 7 to 30 mol%. 63. The LNP or tLNP of any one of claims 54-62, comprising a sterol in an amount in the range from 20 to 45 mol%. 64. The LNP or tLNP of any one of claims 54-63, comprising at least one co-lipid in an amount in the range from 1 to 30 mol%. 65. The LNP or tLNP of any one of claims 54-64, comprising at least one unfunctionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%. 66. The LNP or tLNP of any one of claims 53-65, comprising at least one functionalized PEG-lipid in an amount in the range from 0.1 to 5 mol%. 67. The tLNP of any one of claims 53-66, wherein the binding moiety comprises an antigen, a ligand-binding domain of a receptor, a receptor ligand, an antibody, or an antigen binding domain of an antibody. 68. The LNP of any one of claims 52 or 54-66 or the tLNP of any one of claims 53-67, further comprising a nucleic acid molecule payload. 69. The LNP or tLNP of claim 68, wherein the weight ratio of total lipid to nucleic acid is 10:1 to 50:1. 70. The LNP or tLNP of claim 68, wherein the N/P ratio is from 3 to 9. 71. The LNP or tLNP of any one of claims 68-70, wherein the nucleic acid molecule is an mRNA. 72. The tLNP of any one of claims 68-71, wherein the binding moiety is a whole antibody and the ratio of antibody to nucleic acid molecule is from about 0.3 to about 1.0 (w/w). 73. The tLNP of any one of claims 53-71, wherein the binding moiety is a F(ab’) or F(ab’) analog. 74. A method of delivering a nucleic acid into a cell comprising contacting the cell with the LNP or tLNP any one of claims 68-73.
PCT/US2025/024156 2024-04-11 2025-04-10 Ionizable cationic lipids and lipid nanoparticles Pending WO2025217454A2 (en)

Applications Claiming Priority (10)

Application Number Priority Date Filing Date Title
US202463632940P 2024-04-11 2024-04-11
US63/632,940 2024-04-11
US202463654928P 2024-05-31 2024-05-31
US202463654704P 2024-05-31 2024-05-31
US202463654930P 2024-05-31 2024-05-31
US63/654,704 2024-05-31
US63/654,928 2024-05-31
US202463708461P 2024-10-17 2024-10-17
US202463708529P 2024-10-17 2024-10-17
US63/708,529 2024-10-17

Publications (2)

Publication Number Publication Date
WO2025217454A2 true WO2025217454A2 (en) 2025-10-16
WO2025217454A3 WO2025217454A3 (en) 2025-11-20

Family

ID=95651408

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/024156 Pending WO2025217454A2 (en) 2024-04-11 2025-04-10 Ionizable cationic lipids and lipid nanoparticles

Country Status (1)

Country Link
WO (1) WO2025217454A2 (en)

Citations (429)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989006968A1 (en) 1988-02-03 1989-08-10 Xoma Corporation Therapeutic use of anti-t cell immunotoxin for autoimmune diseases
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO2001023573A1 (en) 1999-09-30 2001-04-05 Kyowa Hakko Kogyo Co., Ltd. Human type complementation-determining domain transplanted antibody against ganglioside gd2 and derivative of this antibody
WO2002030459A1 (en) 2000-10-11 2002-04-18 Tegenero Gmbh Use of cd28-specific monoclonal antibodies for stimulating blood cells that lack cd28
WO2002047721A1 (en) 2000-12-14 2002-06-20 Fujisawa Pharmaceutical Co., Ltd. Silensed anti-cd28 antibodies and use thereof
WO2004045512A2 (en) 2002-11-15 2004-06-03 Genmab A/S Human monoclonal antibodies against cd25
WO2004055056A1 (en) 2002-12-17 2004-07-01 Merck Patent Gmbh Humanized antibody (h14.18) of the mouse 14.18 antibody binding to gd2 and its fusion with il-2
WO2005035584A1 (en) 2003-10-10 2005-04-21 Bristol-Myers Squibb Company Fully human antibodies against human 4-1bb (cd137)
WO2005037999A2 (en) 2003-10-14 2005-04-28 Biogen Idec Ma Inc. Treatment of cancer using antibodies to lrrc15
US6924360B2 (en) 2001-12-28 2005-08-02 Abgenix, Inc. Antibodies against the MUC18 antigen
WO2006108670A2 (en) 2005-04-15 2006-10-19 Novartis Ag Use of cd25 antibodies in immunotherapy
WO2007009065A2 (en) 2005-07-11 2007-01-18 Macrogenics, Inc. Methods of treating autoimmune disease using humanized anti-cd16a antibodies
WO2007084321A2 (en) 2006-01-12 2007-07-26 Alexion Pharmaceuticals, Inc. Antibodies to ox-2/cd200 and uses thereof
US7378504B2 (en) 2004-06-03 2008-05-27 Medarex, Inc. Human monoclonal antibodies to Fc gamma receptor I (CD64)
WO2008079246A2 (en) 2006-12-21 2008-07-03 Medarex, Inc. Cd44 antibodies
WO2008117049A1 (en) 2007-03-26 2008-10-02 Affitech As Human anti cd166 antibody binding human tumor cells
WO2008121160A2 (en) 2006-11-21 2008-10-09 Xencor, Inc. Optimized antibodies that target cd5
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
WO2008144890A1 (en) 2007-05-30 2008-12-04 F. Hoffmann-La Roche Ag Chimeric and humanized anti-cd44 antibodies that mediate cancer cell cytotoxicity
US20100040540A1 (en) 2003-11-19 2010-02-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anti cd44 antibodies for eradicating leukaemic stem cells and breast cancer stem cells
WO2010022737A1 (en) 2008-08-29 2010-03-04 Symphogen A/S Anti-cd5 antibodies
WO2010032059A2 (en) 2008-09-19 2010-03-25 Medimmune Llc Targeted binding agents directed to cd105 and uses thereof
WO2010039873A2 (en) 2008-10-01 2010-04-08 Tracon Pharmaceuticals, Inc. Humanized endoglin antibodies
US7722869B2 (en) 2007-04-27 2010-05-25 Novartis Ag Antibody molecules and nucleic acids
WO2010104949A2 (en) 2009-03-10 2010-09-16 Biogen Idec Ma Inc. Anti-bcma antibodies
US7825222B2 (en) 2001-02-12 2010-11-02 Novartis Ag Therapeutic binding molecules
US7915391B2 (en) 2006-04-24 2011-03-29 Amgen Inc. Humanized c-Kit antibody
WO2011094259A2 (en) 2010-01-28 2011-08-04 Glaxo Group Limited Cd127 binding proteins
WO2011104687A1 (en) 2010-02-24 2011-09-01 Rinat Neuroscience Corporation Antagonist anti-il-7 receptor antibodies and methods
WO2011127324A2 (en) 2010-04-08 2011-10-13 JN Biosciences, LLC Antibodies to cd122
US20110280881A1 (en) 2010-04-13 2011-11-17 Riken Novel anti-hsp90 monoclonal antibody
US8124738B2 (en) 2005-09-26 2012-02-28 Medarex, Inc. Human monoclonal antibodies to CD70
WO2012032433A1 (en) 2010-09-09 2012-03-15 Pfizer Inc. 4-1bb binding molecules
WO2012071216A2 (en) 2010-11-22 2012-05-31 Duke University Antibodies for tumor gangliosides
US8221753B2 (en) 2009-09-30 2012-07-17 Tracon Pharmaceuticals, Inc. Endoglin antibodies
US8236318B2 (en) 2007-11-07 2012-08-07 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US8278036B2 (en) 2005-08-23 2012-10-02 The Trustees Of The University Of Pennsylvania RNA containing modified nucleosides and methods of use thereof
WO2012138537A1 (en) 2011-04-01 2012-10-11 Immunogen, Inc. Compositions and methods for treatment of ovarian, peritoneal, and fallopian tube cancer
WO2012149412A2 (en) 2011-04-29 2012-11-01 Immunogen Inc. Anti-endoglin (cd105) antibodies, immunoconjugates, and uses thereof
WO2012163805A1 (en) 2011-05-27 2012-12-06 Glaxo Group Limited Bcma (cd269/tnfrsf17) -binding proteins
US8383117B2 (en) 2004-06-10 2013-02-26 Viventia Biotechnologies Inc. CD44E tumor specific antibody
WO2013056984A1 (en) 2011-10-19 2013-04-25 Effimune Antibodies directed against the alpha chain of il7 receptor - their use for the preparation of drug candidates
US20130115274A1 (en) 2011-11-04 2013-05-09 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
US8454959B2 (en) 2008-07-18 2013-06-04 Bristol-Meyers Squibb Company Compositions monovalent for CD28 binding and methods of use
US8507657B2 (en) 2008-06-30 2013-08-13 Morphotek, Inc. Anti-GD2 antibodies
US8552157B2 (en) 2011-05-12 2013-10-08 Imclone Llc c-Kit antibodies and uses thereof
WO2013167259A1 (en) 2012-05-09 2013-11-14 Ganymed Pharmaceuticals Ag Antibodies against claudin 18.2 useful in cancer diagnosis
WO2014000338A1 (en) 2012-06-28 2014-01-03 中国科学院生物物理研究所 Application of composite of anti-cd146 antibody and anti-vegf antibody in preparing medicine for suppressing tumor
US8679500B2 (en) 2009-05-14 2014-03-25 Institute National De La Sante Et De La Recherche Medicale (Inserm) Compositions containing antibodies for treating CD5+ HLA-DR+ B or T cell related diseases
US8722858B2 (en) 2007-06-25 2014-05-13 Chugai Seiyaku Kabushiki Kaisha Anti-Prominin-1 antibody having ADCC activity or CDC activity
WO2014083379A1 (en) 2012-11-30 2014-06-05 Institut Pasteur Use of anti-fcyri and/or anti-fcyriia antibodies for treating arthritis, inflammation, thrombocytopenia and allergic shock
US8785604B2 (en) 2010-02-18 2014-07-22 Effimune Anti-CD28 humanized antibodies
WO2014128185A1 (en) 2013-02-21 2014-08-28 Universitätsklinikum Freiburg Recombinant bispecific antibody that binds to the cd133 antigen on tumor cells and to the human cd3 t cell receptor
WO2014158821A1 (en) 2013-03-12 2014-10-02 Imaginab, Inc. Antigen binding constructs to cd70
US20140308301A1 (en) 2011-10-26 2014-10-16 The Regents Of The University Of California Cd44 monoclonal antibody for the treatment of b-cell chronic lymphocytic leukemia and other hematological malignancies
US8951524B2 (en) 2006-10-10 2015-02-10 Universite De Nantes Use of monoclonal antibodies specific to the O-acetylated form of GD2 ganglioside for treatment of certain cancers
WO2015052538A1 (en) 2013-10-10 2015-04-16 Ucl Business Plc Chimeric antigen receptor
WO2015076425A1 (en) 2013-11-25 2015-05-28 リンク・ジェノミクス株式会社 New monoclonal antibody
WO2015118031A2 (en) 2014-02-06 2015-08-13 Oncomatryx Biopharma, S.L. Antibody-drug conjugates and immunotoxins
US9115192B2 (en) 2010-09-27 2015-08-25 Zestagen S.A. Compositions and methods for treating neoplasia
WO2015132604A1 (en) 2014-03-06 2015-09-11 Ucl Business Plc Chimeric antigen receptor
WO2015140591A1 (en) 2014-03-21 2015-09-24 Nordlandssykehuset Hf Anti-cd14 antibodies and uses thereof
US20150337039A1 (en) 2014-05-22 2015-11-26 Case Western Reserve University Mac-1 antibodies and uses thereof
WO2015184203A1 (en) 2014-05-29 2015-12-03 Macrogenics, Inc. Tri-specific binding molecules and methods of use thereof
WO2015187528A1 (en) 2014-06-02 2015-12-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting cd-19
WO2015189302A1 (en) 2014-06-10 2015-12-17 Effimune Antibodies directed against cd127
US9220772B2 (en) 2010-07-22 2015-12-29 The Regents Of The University Of California Anti-tumor antigen antibodies and methods of use
US9243058B2 (en) 2012-12-07 2016-01-26 Amgen, Inc. BCMA antigen binding proteins
US9249225B2 (en) 2010-05-26 2016-02-02 Regents Of The University Of Minnesota Single chain variable fragment anti-CD133 antibodies and uses thereof
US9273141B2 (en) 2011-05-27 2016-03-01 Glaxo Group Limited B cell maturation antigen (BCMA) binding proteins
US9284375B2 (en) 2005-04-15 2016-03-15 Macrogenics, Inc. Covalent diabodies and uses thereof
US9315585B2 (en) 2010-06-19 2016-04-19 Memorial Sloan Kettering Cancer Center Anti-GD2 antibodies
US9328156B2 (en) 2010-12-09 2016-05-03 The Trustees Of The University Of Pennsylvania Use of chimeric antigen receptor-modified T cells to treat cancer
WO2016090337A1 (en) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Chimeric antigen receptors targeting fc receptor-like 5 and uses thereof
WO2016090329A2 (en) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Antibodies targeting g-protein coupled receptor and methods of use
US9382321B2 (en) 2014-11-26 2016-07-05 Adventis Health System/Sunbelt, Inc. Effector-deficient anti-CD32A antibodies
US9388249B2 (en) 2006-07-10 2016-07-12 Amano Enzyme Inc. Method of classifying antibody, method of identifying antigen, method of obtaining antibody or antibody set, method of constructing antibody panel and antibody or antibody set and use of the same
WO2016130598A1 (en) 2015-02-09 2016-08-18 University Of Florida Research Foundation, Inc. Bi-specific chimeric antigen receptor and uses thereof
US9447190B2 (en) 2012-09-10 2016-09-20 Prothena Biosciences Limited Anti-MCAM antibodies and associated methods of use
WO2016149578A1 (en) 2015-03-19 2016-09-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Dual specific anti-cd22-anti-cd19 chimeric antigen receptors
WO2016166630A1 (en) 2015-04-13 2016-10-20 Pfizer Inc. Chimeric antigen receptors targeting b-cell maturation antigen
US9493740B2 (en) 2010-09-08 2016-11-15 Baylor College Of Medicine Immunotherapy of cancer using genetically engineered GD2-specific T cells
WO2016187216A1 (en) 2015-05-18 2016-11-24 Bluebird Bio, Inc. Anti-ror1 chimeric antigen receptors
WO2016197974A1 (en) 2015-06-12 2016-12-15 Yen-Ta Lu Methods and antibodies for modulation of immunoresponse
US9540443B2 (en) 2011-01-26 2017-01-10 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies
WO2017009473A1 (en) 2015-07-16 2017-01-19 Ucb Biopharma Sprl Antibody molecules which bind cd45
WO2017021540A1 (en) 2015-08-06 2017-02-09 Agency For Science, Technology And Research Il2rbeta/common gamma chain antibodies
US20170051068A1 (en) 2015-08-17 2017-02-23 Janssen Pharmaceutica Nv Anti-BCMA Antibodies, Bispecific Antigen Binding Molecules that Bind BCMA and CD3, and Uses Thereof
WO2017048770A1 (en) 2015-09-15 2017-03-23 Regulus Therapeutics, Inc. Systems, compositions, and methods for formulating nucleic acid compositions
WO2017055385A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xgd2 bispecific t cell activating antigen binding molecules
WO2017058866A1 (en) 2015-09-28 2017-04-06 Precision Immune, Inc. Anti-trem2 antibodies and uses thereof
US9617349B2 (en) 2004-01-22 2017-04-11 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
WO2017062748A1 (en) 2015-10-07 2017-04-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Il-7r-alpha specific antibodies for treating acute lymphoblastic leukemia
WO2017064221A1 (en) 2015-10-13 2017-04-20 Affimed Gmbh Multivalent fv antibodies
WO2017096120A1 (en) 2015-12-04 2017-06-08 Memorial Sloan-Kettering Cancer Center Antibodies targeting fc receptor-like 5 and methods of use
US20170166638A1 (en) 2014-03-26 2017-06-15 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. USE OF CD-64-BLOCKING AGENTS AS ENHANCER OF AN ANTI-TNF-a ANTIBODY THERAPY
US9688755B2 (en) 2007-05-14 2017-06-27 Novimmune S.A. Fc receptor-binding polypeptides with modified effector functions
US9701752B2 (en) 2005-04-19 2017-07-11 Seattle Genetics, Inc. Humanized anti-CD70 binding agents and uses thereof
US9701756B2 (en) 2014-11-26 2017-07-11 Miltenyi Biotec Gmbh Humanized antibody or fragment thereof specific for CD45R0
WO2017118613A1 (en) 2016-01-08 2017-07-13 Syddansk Universitet Bispecific antibodies targeting human cd73
WO2017123650A2 (en) 2016-01-11 2017-07-20 Inhibrx Lp Multivalent and multispecific 41bb-binding fusion proteins
WO2017130223A2 (en) 2016-01-29 2017-08-03 Virocan Therapeutics Pvt. Ltd. A chimeric antigen receptor specific to b-cell maturation antigen, a recombinant expression vector and a method thereof
WO2017134306A1 (en) 2016-02-05 2017-08-10 Orionis Biosciences Nv Cd8 binding agents
WO2017156479A1 (en) 2016-03-11 2017-09-14 Bluebird Bio, Inc. Ror1 chimeric antigen receptors
US9765148B2 (en) 2011-03-16 2017-09-19 Argen-X N.V. Antibodies to CD70
US9777068B2 (en) 2012-06-18 2017-10-03 Apeiron Biologics Ag Method for treating a GD2 positive cancer
WO2017173256A1 (en) 2016-04-01 2017-10-05 Kite Pharma, Inc. Chimeric antigen and t cell receptors and methods of use
US9782500B2 (en) 2013-09-24 2017-10-10 Oncoinvent As Monoclonal antibody and derivatives
US9789203B2 (en) 2013-03-15 2017-10-17 Novartis Ag cKIT antibody drug conjugates
US20170335016A1 (en) 2008-04-25 2017-11-23 Kyowa Hakko Kirin Co., Ltd Stable multivalent antibody
WO2017213979A1 (en) 2016-06-06 2017-12-14 St. Jude Children's Research Hospital Anti-cd7 chimeric antigen receptor and method of use thereof
US9856324B2 (en) 2014-06-04 2018-01-02 MabVax Therapeutics, Inc. Human monoclonal antibodies to ganglioside GD2
WO2018006882A1 (en) 2016-07-08 2018-01-11 科济生物医药（上海）有限公司 Antibody for anti-claudin 18a2 and use thereof
WO2018010846A1 (en) 2016-07-15 2018-01-18 Ogd2 Pharma Humanized antibody against o-acetylated gd2 ganglioside (oacgd2)
WO2018017786A2 (en) 2016-07-20 2018-01-25 Janssen Pharmaceutica Nv Anti- gprc5d antibodies, bispecific antigen binding molecules that bind gprc5d and cd3, and uses thereof
WO2018028647A1 (en) 2015-08-11 2018-02-15 Legend Biotech Usa Inc. Chimeric antigen receptors targeting bcma and methods of use thereof
WO2018039626A1 (en) 2016-08-25 2018-03-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Human single domain antibodies targeting cd16a to mediate adcc
WO2018045880A1 (en) 2016-09-06 2018-03-15 Guangzhou Bainifu Biotech Co., Ltd Chimeric antigen receptor, car133-nkt cells and use thereof
US9926375B2 (en) 2014-11-12 2018-03-27 Tracon Pharmaceuticals, Inc. Anti-endoglin antibodies and uses thereof
US9932410B2 (en) 2013-11-05 2018-04-03 Inserm (Institut National De La Sante Et De La Recherche Medicale Human neutralizing anti-kit antibodies and uses thereof
US9938356B2 (en) 2014-11-10 2018-04-10 Medimmune Limited Binding molecules specific for CD73 and uses thereof
WO2018072025A1 (en) 2016-10-19 2018-04-26 The Governing Council Of The University Of Toronto Cd133-binding agents and uses thereof
WO2018098306A1 (en) 2016-11-22 2018-05-31 National University Of Singapore Blockade of cd7 expression and chimeric antigen receptors for immunotherapy of t-cell malignancies
WO2018147245A1 (en) 2017-02-07 2018-08-16 第一三共株式会社 Anti-gprc5d antibody and molecule containing same
WO2018152516A1 (en) 2017-02-20 2018-08-23 Adimab, Llc Proteins binding cd33, nkg2d and cd16
WO2018158349A1 (en) 2017-02-28 2018-09-07 Affimed Gmbh Tandem diabody for cd16a-directed nk-cell engagement
US10081682B2 (en) 2013-10-11 2018-09-25 Oxford Bio Therapeutics Ltd. Conjugated antibodies against LY75 for the treatment of cancer
US10100129B2 (en) 2014-11-21 2018-10-16 Bristol-Myers Squibb Company Antibodies against CD73 and uses thereof
US10106623B2 (en) 2014-02-12 2018-10-23 Michael Uhlin Bispecific antibodies for use in stem cell transplantation
US10108123B2 (en) 2016-09-29 2018-10-23 Canon Kabushiki Kaisha Image forming apparatus
US10106609B2 (en) 2014-06-04 2018-10-23 PeronGen BioTherapeutics (Suzhou) Co., Ltd. CD7 nanobodies, encoding sequence and use thereof
WO2018191786A1 (en) 2017-04-21 2018-10-25 Implicit Bioscience Pty Ltd Cd 14 antagonist antibodies for treating neurodegenerative diseases
WO2018197675A1 (en) 2017-04-28 2018-11-01 Julius-Maximilians-Universität Würzburg Ror1-specific chimeric antigen receptors (car) with humanized targeting domains
WO2018199337A1 (en) 2017-04-28 2018-11-01 味の素株式会社 Compound having substance that has affinity for soluble protein, cleavable moiety, and reactive group, or salt thereof
WO2018213337A1 (en) 2017-05-15 2018-11-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Bicistronic chimeric antigen receptors and their uses
WO2018220467A1 (en) 2017-05-30 2018-12-06 Foroogh Nejatollahi Anti-muc18 human immunotoxin and applications thereof
WO2018235247A1 (en) 2017-06-22 2018-12-27 学校法人順天堂 ANTI-CD10 ANTIBODIES
WO2019006072A1 (en) 2017-06-30 2019-01-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-b-cell maturation antigen chimeric antigen receptors with human domains
US10174122B2 (en) 2017-01-06 2019-01-08 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
WO2019032661A1 (en) 2017-08-09 2019-02-14 Orionis Biosciences Inc. Cd8 binding agents
WO2019033043A2 (en) 2017-08-11 2019-02-14 Genentech, Inc. Anti-cd8 antibodies and uses thereof
WO2019059771A2 (en) 2017-09-21 2019-03-28 Umc Utrecht Holding B.V. Anti-gd2 antibody for the treatment of neuroblastoma
US10266604B2 (en) 2011-09-22 2019-04-23 Amgen Inc. CD27L antigen binding proteins
US10273294B2 (en) 2010-10-11 2019-04-30 University Of Southern California Compositions and methods for treating HIF-1α over-expressing cancers
US10273295B2 (en) 2014-08-19 2019-04-30 Miltenyi Biotec Gmbh Chimeric antigen receptor specific for SSEA4 antigen
WO2019084067A1 (en) 2017-10-24 2019-05-02 Magenta Therapeutics, Inc. Anti-cd117 antibodies and methods of using same
WO2019085102A1 (en) 2017-11-03 2019-05-09 西安宇繁生物科技有限责任公司 Bcma-specific chimeric antigen receptor t cell and application thereof
WO2019108863A1 (en) 2017-11-29 2019-06-06 Magenta Therapeutics, Inc. Compositions and methods for the depletion of cd5+ cells
US10316101B2 (en) 2014-04-14 2019-06-11 Cellectis BCMA (CD269) specific chimeric antigen receptors for cancer immunotherapy
WO2019119822A1 (en) 2017-12-23 2019-06-27 Uwell Biopharma Inc. Pharmaceutical chimeric receptor composition and method thereof
WO2019126536A1 (en) 2017-12-20 2019-06-27 Alexion Pharmaceuticals Inc. Humanized anti-cd200 antibodies and uses thereof
WO2019133639A2 (en) 2017-12-26 2019-07-04 The Regents Of The University Of California Human antibodies that bind and are internalized by mesothelioma and other cancer cells
WO2019137309A1 (en) 2018-01-09 2019-07-18 南京任诺药业有限公司 Humanized anti-human cd146 monoclonal antibody having high neutralizing activity
WO2019154890A1 (en) 2018-02-09 2019-08-15 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
US10383929B2 (en) 2014-12-12 2019-08-20 Bluebird Bio, Inc. BCMA chimeric antigen receptors
US10385137B2 (en) 2004-12-16 2019-08-20 Inserm (Institut National De La Sante Et De La Recherche Medicale) Production of antibody formats and immunological applications of said formats
WO2019159193A1 (en) 2018-02-13 2019-08-22 Indian Institute Of Technology Bombay Novel humanized anti-cd19 chimeric antigen receptor, its nucelic acid sequence and its preparation
US10407506B2 (en) 2014-03-12 2019-09-10 Prothena Biosciences Limited Anti-MCAM antibodies and associated methods of use
US10406179B2 (en) 2014-08-26 2019-09-10 The Board Of Trustees Of The Leland Stanford Junior University Engraftment of stem cells with a combination of an agent that targets stem cells and modulation of immunoregulatory signaling
WO2019170131A1 (en) 2018-03-07 2019-09-12 复旦大学 Targeted cd73 antibody and antibody-drug conjugate, and preparation method therefor and uses thereof
WO2019173692A2 (en) 2018-03-09 2019-09-12 Agenus Inc. Anti-cd73 antibodies and methods of use thereof
US10414820B2 (en) 2013-03-13 2019-09-17 Imaginab, Inc. Antigen binding constructs to CD8
WO2019175226A1 (en) 2018-03-13 2019-09-19 Tusk Therapeutics Ltd Anti-cd25 antibody agents
US10426797B2 (en) 2017-03-24 2019-10-01 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-CD33 immunotherapy
US10428141B2 (en) 2017-11-03 2019-10-01 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-ROR1 immunotherapy
WO2019190952A1 (en) 2018-03-29 2019-10-03 Lan Bo Chen Monoclonal antibodies that bind to ssea4 and uses thereof
US10442867B2 (en) 2017-07-31 2019-10-15 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-CD19/CD20 immunotherapy
US20190321404A1 (en) 2016-12-30 2019-10-24 Nanjing Legend Biotech Co., Ltd. Novel chimeric antigen receptor and use thereof
US10457726B2 (en) 2016-06-30 2019-10-29 University Of Connecticut Antibody and antigen-binding fragment compositions targeting cell surface antigens in tumors and methods of use thereof
WO2019219089A1 (en) 2018-05-18 2019-11-21 Bridge Health Bio-Tech Co., Ltd Anti-claudin 18.2 antibodies and uses thereof
US10508148B2 (en) 2017-12-12 2019-12-17 Pionyr Immunotherapeutics, Inc. Anti-TREM2 antibodies and related methods
WO2019241426A1 (en) 2018-06-13 2019-12-19 Novartis Ag Bcma chimeric antigen receptors and uses thereof
WO2019240287A1 (en) 2018-06-14 2019-12-19 味の素株式会社 Compound comprising substance having affinity for antibody, cleavage site and reactive group, or salt thereof
WO2020014366A1 (en) 2018-07-10 2020-01-16 Intrexon Corporation Ror-1 specific chimeric antigen receptors and uses thereof
WO2020011706A1 (en) 2018-07-09 2020-01-16 Oslo Universitetssykehus Hf Two chimeric antigen receptors specifically binding cd19 and igkappa
US10543263B2 (en) 2017-10-16 2020-01-28 Lentigen Technology Inc. Compositions and methods for treating cancer with anti-CD22 immunotherapy
WO2020020194A1 (en) 2018-07-24 2020-01-30 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Gd2-based chimeric antigen receptor and application thereof
US10548987B2 (en) 2015-07-31 2020-02-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody-drug conjugates for targeting CD56-positive tumors
US20200040101A1 (en) 2018-07-18 2020-02-06 Askgene Pharma Inc. Novel antibodies and methods for making and using the same
WO2020025792A1 (en) 2018-08-03 2020-02-06 Amgen Research (Munich) Gmbh Antibody constructs for cldn18.2 and cd3
WO2020038146A1 (en) 2018-08-24 2020-02-27 深圳普瑞金生物药业有限公司 Bcma chimeric antigen receptor based on single domain antibody and use thereof
US10584169B2 (en) 2017-01-24 2020-03-10 I-Mab Biopharma Us Limited Anti-CD73 antibodies and uses thereof
WO2020055975A1 (en) 2018-09-11 2020-03-19 Washington University Anti-trem-2 agonist antibodies
WO2020060924A1 (en) 2018-09-17 2020-03-26 Dualogics, Llc Use of a cd4/cd8 bispecific antibody for the treatment of autoimmune/inflammatory disorders
WO2020065330A2 (en) 2018-09-27 2020-04-02 Autolus Limited Chimeric antigen receptor
US10611838B2 (en) 2018-02-09 2020-04-07 Ultrahuman Five Limited C-KIT antibodies
WO2020076105A1 (en) 2018-10-10 2020-04-16 주식회사 노벨티노빌리티 Novel anti-c-kit antibody
US10633444B2 (en) 2016-04-18 2020-04-28 Celldex Therapeutics, Inc. Agonistic antibodies that bind CD40
WO2020092854A2 (en) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Chimeric antigen receptors specific for g protein-coupled receptor class c group 5 member d (gprc5d)
WO2020092654A1 (en) 2018-10-30 2020-05-07 Magenta Therapeutics, Inc. Anti-cd45 antibodies and conjugates thereof
WO2020098599A1 (en) 2018-11-12 2020-05-22 江苏恒瑞医药股份有限公司 Anti-cd73 antibody, antigen-binding fragment thereof and application thereof
WO2020102591A1 (en) 2018-11-14 2020-05-22 Rubryc Therapeutics, Inc. Cd25 antibodies
US10676525B2 (en) 2017-08-03 2020-06-09 Alector Llc Anti-TREM2 antibodies and methods of use thereof
US20200181260A1 (en) 2017-02-22 2020-06-11 H. Lee Moffitt Cancer Center And Research Institute, Inc. Bispecific antibody for cancer immunotherapy
US10683369B2 (en) 2015-08-03 2020-06-16 Engmab Sàrl Monoclonal antibodies against BCMA
WO2020121195A1 (en) 2018-12-10 2020-06-18 Mor Research Applications Trem2 antibodies and uses thereof
US10689456B2 (en) 2014-12-08 2020-06-23 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-CD70 chimeric antigen receptors
WO2020127618A1 (en) 2018-12-21 2020-06-25 F. Hoffmann-La Roche Ag Tumor-targeted agonistic cd28 antigen binding molecules
WO2020132190A1 (en) 2018-12-21 2020-06-25 Multitude Inc. Antibodies specific to muc18
WO2020135201A1 (en) 2018-12-28 2020-07-02 四川科伦博泰生物医药股份有限公司 Antibody and use thereof
WO2020139956A1 (en) 2018-12-28 2020-07-02 Sparx Therapeutics Inc. Binding molecules specific for claudin 18.2, compositons and methods thereof, for treatment of cancer and other diseases
US10711068B2 (en) 2015-03-26 2020-07-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-CD133 monoclonal antibodies and related compositions and methods
WO2020143836A1 (en) 2019-01-11 2020-07-16 上海开拓者生物医药有限公司 Cd73 antibody, preparation method therefor and application thereof
WO2020143710A1 (en) 2019-01-11 2020-07-16 康诺亚生物医药科技（成都）有限公司 Anti-cd73 monoclonal antibody and application thereof
US10716851B2 (en) 2017-07-11 2020-07-21 Compass Therapeutics Llc Agonist antibodies that bind human CD137 and uses thereof
WO2020147321A1 (en) 2019-01-17 2020-07-23 Beijing Mabworks Biotech Co. Ltd. Antibodies binding human claudin 18.2 and uses thereof
WO2020148677A1 (en) 2019-01-18 2020-07-23 Janssen Biotech, Inc. Gprc5d chimeric antigen receptors and cells expressing the same
WO2020154293A1 (en) 2019-01-22 2020-07-30 Bristol-Myers Squibb Company Antibodies against il-7r alpha subunit and uses thereof
US10730941B2 (en) 2015-07-31 2020-08-04 Memorial Sloan-Kettering Cancer Center Antigen-binding proteins targeting CD56 and uses thereof
US10730944B2 (en) 2017-07-24 2020-08-04 Regeneron Pharmaceuticals, Inc. Anti-CD8 antibodies and uses thereof
WO2020160242A1 (en) 2019-02-01 2020-08-06 Regeneron Pharmaceuticals, Inc. Anti-il2 receptor gamma antigen-binding proteins
US20200246381A1 (en) 2018-02-01 2020-08-06 Nanjing Iaso Biotherapeutics Co., Ltd. Chimeric antigen receptor (car) binding to bcma, and uses thereof
WO2020159754A2 (en) 2019-01-28 2020-08-06 Multitude Inc. Antibodies specific to cd44
US10738121B2 (en) 2015-02-27 2020-08-11 The General Hospital Corporation Therapeutic use of integrin-binding antibodies
US10745481B2 (en) 2015-05-04 2020-08-18 Cytomx Therapeutics, Inc. Anti-CD166 antibodies, activatable anti-CD166 antibodies, and methods of use thereof
US20200261503A1 (en) 2018-12-01 2020-08-20 Allogene Therapeutics, Inc. Chimeric antigen receptors targeting b-cell maturation antigen and methods of use thereof
US10752684B2 (en) 2014-07-29 2020-08-25 Cellectis ROR1 (NTRKR1) specific chimeric antigen receptors for cancer immunotherapy
US10758625B2 (en) 2015-04-14 2020-09-01 Benhealth Biopharmaceutic (Shenzhen) Co., Ltd. Bispecific antibody capable of being combined with immune cells to enhance tumor killing capability, and preparation method therefor and application thereof
WO2020184944A1 (en) 2019-03-08 2020-09-17 앱티스 주식회사 Site-specific antibody conjugation and antibody-drug conjugate as specific example thereof
WO2020190737A1 (en) 2019-03-15 2020-09-24 Cartesian Therapeutics, Inc. Anti-bcma chimeric antigen receptors
US20200297634A1 (en) 2019-03-19 2020-09-24 Arcturus Therapeutics, Inc. Method of making lipid-encapsulated rna nanoparticles
WO2020198531A2 (en) 2019-03-26 2020-10-01 The Regents Of The University Of California Chimeric antigen receptor modified t-cells (car-t) for the treatment of hematological and solid tumor cancers
WO2020212710A1 (en) 2019-04-18 2020-10-22 Kymab Limited Antagonists anti-cd7 antibodies
US20200339699A1 (en) 2018-02-01 2020-10-29 Innovent Biologics (Suzhou) Co., Ltd. Fully humanized anti-b cell maturation antigen (bcma) single-chain antibody and use thereof
WO2020219770A1 (en) 2019-04-24 2020-10-29 Magenta Therapeutics, Inc. Anti-cd117 antibodies and uses thereof
WO2020216697A1 (en) 2019-04-23 2020-10-29 Innate Pharma Cd73 blocking antibodies
US10822426B2 (en) 2015-12-09 2020-11-03 Corvus Pharmaceuticals, Inc. Humanized anti-CD73 antibodies
US10844128B2 (en) 2018-09-20 2020-11-24 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-CD123 immunotherapy
WO2020253568A1 (en) 2019-06-19 2020-12-24 海正生物制药有限公司 Anti-cd73 antibody and application thereof
WO2020254827A1 (en) 2019-06-21 2020-12-24 Vhsquared Limited Polypeptides
US20210002384A1 (en) 2018-02-15 2021-01-07 St. Jude Children's Research Hospital Process for producing hu14.18k322a monoclonal antibody
WO2021008463A1 (en) 2019-07-12 2021-01-21 明济生物制药（北京）有限公司 Cldn18.2 antibody and use thereof
WO2021018925A1 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021017892A1 (en) 2019-07-26 2021-02-04 上海复宏汉霖生物技术股份有限公司 Method and composition for anti-cd73 antibodies and variants
WO2021022304A2 (en) 2019-07-30 2021-02-04 Qlsf Biotherapeutics, Inc. MULTISPECIFIC BINDING COMPOUNDS THAT BIND TO LRRC15 AND CD3ε
WO2021018859A2 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
US10919967B2 (en) 2016-06-21 2021-02-16 Albert-Ludwigs-Universitätfreiburg Antibodies against Mac-1
US10920246B2 (en) 2015-05-26 2021-02-16 Ramot At Tel-Aviv University Ltd. Targeted lipid particles for systemic delivery of nucleic acid molecules to leukocytes
WO2021027850A1 (en) 2019-08-12 2021-02-18 I-Mab Biopharma Co., Ltd. Anti-claudin 18.2 and anti-4-1bb bispecific antibodies and uses thereof
WO2021032173A1 (en) 2019-08-21 2021-02-25 Harbour Biomed (Shanghai) Co., Ltd. Anti-cd73 antibody and application thereof
WO2021032157A1 (en) 2019-08-20 2021-02-25 Mabspace Biosciences (Suzhou) Co., Limited Novel anti-cldn18.2 antibodies
WO2021044039A1 (en) 2019-09-06 2021-03-11 Scancell Limited Ssea-4 binding members
WO2021044008A1 (en) 2019-09-04 2021-03-11 Universität Zürich Bispecific binding agent that binds to CD117/c-KIT and CD3
WO2021046159A1 (en) 2019-09-04 2021-03-11 Genentech, Inc. Cd8 binding agents and uses thereof
US20210087295A1 (en) 2019-09-23 2021-03-25 The Trustees Of The University Of Pennsylvania Disrupting tumor tissues by targeting fibroblast activation protein (fap)
WO2021055437A1 (en) 2019-09-16 2021-03-25 The General Hospital Corporation Cd70 targeted chimeric antigen receptor (car) t cells and uses thereof
WO2021072312A1 (en) 2019-10-11 2021-04-15 The Trustees Of The University Of Pennsylvania Compositions and methods for targeting cd13 and tim-3 with car t cells to treat acute myeloid leukemia
WO2021076564A1 (en) 2019-10-15 2021-04-22 Dragonfly Therapeutics, Inc. Proteins binding nkg2d, cd16 and flt3
US10988546B2 (en) 2017-08-01 2021-04-27 Medimmune, Llc BCMA monoclonal antibody-drug conjugate
US20210130483A1 (en) 2018-03-26 2021-05-06 Sutro Biopharma, Inc. Anti-bcma receptor antibodies, compositions comprising anti bcma receptor antibodies and methods of making and using anti-bcma antibodies
WO2021091945A1 (en) 2019-11-05 2021-05-14 Janssen Biotech, Inc. Bcma-targeted car-t cell therapy of multiple myeloma
WO2021097223A2 (en) 2019-11-15 2021-05-20 Genzyme Corporation Biparatopic cd73 antibodies
WO2021099418A1 (en) 2019-11-19 2021-05-27 International - Drug - Development - Biotech Anti-cd117 antibodies and methods of use thereof
WO2021101823A1 (en) 2019-11-22 2021-05-27 Eli Lilly And Company Trem2 antibodies and uses thereof
WO2021102332A1 (en) 2019-11-22 2021-05-27 Silverback Therapeutics, Inc. Tgfbetar2 inhibitor-lrrc15 antibody conjugates and uses thereof
WO2021107566A1 (en) 2019-11-25 2021-06-03 주식회사 노벨티노빌리티 Antibody against c-kit and use thereof
US11026975B2 (en) 2018-02-01 2021-06-08 Nanjing Iaso Biotherapeutics Co., Ltd. Chimeric antigen receptor (CAR) binding to BCMA, and uses thereof
WO2021111003A1 (en) 2019-12-06 2021-06-10 SOTIO a.s. Humanized cldn18.2 antibodies
US11034771B2 (en) 2018-07-25 2021-06-15 I-Mab Biopharma Us Limited Anti-CD73 anti-PD-L1 bispecific antibodies
WO2021118955A1 (en) 2019-12-13 2021-06-17 Aggamin Llc Methods and systems for treating or preventing pregnancy-related hypertensive disorders
WO2021127088A1 (en) 2019-12-18 2021-06-24 Janssen Biotech, Inc. Materials and methods for in vivo biological targeting
US11046775B2 (en) 2017-02-14 2021-06-29 Kite Pharma, Inc. CD70 binding molecules and methods of use thereof
WO2021129765A1 (en) 2019-12-27 2021-07-01 Nanjing Legend Biotech Co., Ltd. Claudin18.2 binding moieties and uses thereof
WO2021138378A1 (en) 2019-12-30 2021-07-08 Obi Pharma, Inc. Antibodies, pharmaceutical compositions and uses thereof
WO2021138467A1 (en) 2020-01-03 2021-07-08 Incyte Corporation Anti-cd73 antibodies and uses thereof
WO2021138264A1 (en) 2019-12-30 2021-07-08 Seagen Inc. Methods of treating cancer with nonfucosylated anti-cd70 antibodies
US11078291B2 (en) 2016-02-17 2021-08-03 Seagen Inc. BCMA antibodies and use of same to treat cancer and immunological disorders
WO2021155071A1 (en) 2020-01-29 2021-08-05 Inhibrx, Inc. Cd28 single domain antibodies and multivalent and multispecific constructs thereof
WO2021160269A1 (en) 2020-02-13 2021-08-19 UCB Biopharma SRL Anti cd44-ctla4 bispecific antibodies
WO2021160267A1 (en) 2020-02-13 2021-08-19 UCB Biopharma SRL Bispecific antibodies against cd9 and cd7
WO2021174198A1 (en) 2020-02-28 2021-09-02 The Brigham And Women's Hospital, Inc. Selective modulation of transforming growth factor beta superfamily signaling via multi-specific antibodies
WO2021178896A1 (en) 2020-03-06 2021-09-10 Go Therapeutics, Inc. Anti-glyco-cd44 antibodies and their uses
US11124567B2 (en) 2020-01-13 2021-09-21 Denali Therapeutics Inc. Anti-TREM2 antibodies and methods of use thereof
WO2021188590A2 (en) 2020-03-16 2021-09-23 Magenta Therapeutics, Inc. T-cell bispecific binding proteins
WO2021186056A1 (en) 2020-03-20 2021-09-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Chimeric antigen receptor specific for human cd45rc and uses thereof
US11136403B2 (en) 2017-10-13 2021-10-05 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
WO2021202642A2 (en) 2020-04-01 2021-10-07 The Regents Of The University Of California Immunotheranostic agent targeting mesenchymal stem cell-derived cancer cells and mesenchymal stem cell associated disease
WO2021205383A1 (en) 2020-04-09 2021-10-14 Aprilbio Co., Ltd. Monoclonal antibodies and antigen binding fragments thereof for suppressing cd73 immune checkpoint and uses thereof
WO2021213466A1 (en) 2020-04-22 2021-10-28 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
WO2021222227A1 (en) 2020-04-27 2021-11-04 Memorial Sloan-Kettering Cancer Center Chimeric antigen receptors targeting cd127 and use thereof
WO2021218874A1 (en) 2020-04-27 2021-11-04 启愈生物技术(上海)有限公司 Bispecific antibody targeting human claudin and human pdl1 proteins, and application thereof
US11174319B2 (en) 2019-06-06 2021-11-16 Jacobio Pharmaceuticals Co., Ltd. Binding molecule specific for CD73 and use of binding molecule
WO2021227307A1 (en) 2020-05-12 2021-11-18 普米斯生物技术(珠海)有限公司 Anti-cd73 antibody and use thereof
US11180554B2 (en) 2016-12-13 2021-11-23 Astellas Pharma Inc. Anti-human CD73 antibody
US20210363245A1 (en) 2018-09-17 2021-11-25 The United States Of America,As Represented By The Secretary,Department Of Health And Human Services Bicistronic chimeric antigen receptors targeting cd19 and cd20 and their uses
US11186636B2 (en) 2017-04-21 2021-11-30 Amgen Inc. Anti-human TREM2 antibodies and uses thereof
WO2021241729A1 (en) 2020-05-29 2021-12-02 ブライトパス・バイオ株式会社 Anti-cd73 antibody and use thereof
WO2021245603A1 (en) 2020-06-04 2021-12-09 Crispr Therapeutics Ag Anti-cd70 antibodies and uses thereof
US11203643B2 (en) 2019-05-10 2021-12-21 Lyvgen Biopharma Holdings Limited Humanized anti-CD137 antibodies and uses thereof
WO2021254481A1 (en) 2020-06-19 2021-12-23 信达生物制药(苏州)有限公司 Anti-claudin18.2 antibody and use thereof
US11208482B2 (en) 2018-11-26 2021-12-28 Forty Seven, Inc. Humanized antibodies against c-Kit
WO2021259199A1 (en) 2020-06-22 2021-12-30 信达生物制药(苏州)有限公司 Anti-cd73 antibody and use thereof
WO2022002019A1 (en) 2020-06-30 2022-01-06 江苏恒瑞医药股份有限公司 Anti-cd70 antibody and application thereof
US11220544B2 (en) 2017-07-14 2022-01-11 Cytomx Therapeutics, Inc. Anti-CD166 antibodies and uses thereof
WO2022007808A1 (en) 2020-07-06 2022-01-13 康诺亚生物医药科技（成都）有限公司 Antibody binding to claudin-18.2 and use thereof
WO2022022718A1 (en) 2020-07-31 2022-02-03 北京市神经外科研究所 Anti-cd133 single-chain antibody and use thereof in preparation of drug for treating tumor
WO2022022749A1 (en) 2020-12-16 2022-02-03 南通大学 Use of cd44 antibody in preparation of medicine for treating parkinson's disease
WO2022022720A1 (en) 2020-07-31 2022-02-03 北京市神经外科研究所 Anti-cd44 single-chain antibody and use thereof in preparing drug for treating tumor
US11242385B2 (en) 2017-04-13 2022-02-08 Agenus Inc. Anti-CD137 antibodies and methods of use thereof
US11242395B2 (en) 2017-08-21 2022-02-08 Adagene Inc. Anti-CD137 molecules and use thereof
WO2022032293A2 (en) 2020-08-05 2022-02-10 Vigil Neuroscience, Inc. Treatment of diseases related to colony-stimulating factor 1 receptor dysfunction using trem2 agonists
US11248058B2 (en) 2015-02-19 2022-02-15 University Of Florida Research Foundation, Inc. Chimeric antigen receptors and uses thereof
WO2022040608A1 (en) 2020-08-21 2022-02-24 City Of Hope Anti-cd5 antibody compositions and uses thereof
WO2022040088A2 (en) 2020-08-21 2022-02-24 Synthekine, Inc. Cd45 binding molecules and methods of use
WO2022037531A1 (en) 2020-08-17 2022-02-24 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
WO2022040577A1 (en) 2020-08-20 2022-02-24 City Of Hope Compositions and uses of cd45 targeted chimeric antigen receptor modified t cells
WO2022056199A1 (en) 2020-09-11 2022-03-17 Janssen Biotech, Inc. Multi-specific immune targeting molecules and uses thereof
WO2022064191A1 (en) 2020-09-25 2022-03-31 Ucl Business Ltd Anti-cd45 antibodies and related therapeutics
WO2022063853A1 (en) 2020-09-25 2022-03-31 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates and uses thereof
US11292846B2 (en) 2017-11-02 2022-04-05 Bayer Aktiengesellschaft Bispecific antibodies binding ALK-1 and BMPR-2
WO2022067394A1 (en) 2020-09-30 2022-04-07 Newsouth Innovations Pty Limited Antigen-binding fragments and uses thereof
WO2022068854A1 (en) 2020-09-30 2022-04-07 Nanjing GenScript Biotech Co., Ltd. Antibodies targeting human claudin 18.2 and uses thereof
US11299550B2 (en) 2018-03-09 2022-04-12 Phanes Therapeutics, Inc. Anti-CD73 antibodies and uses thereof
US11306145B2 (en) 2016-04-29 2022-04-19 Viela Bio, Inc. Binding molecules specific for FcγRIIA and uses thereof
WO2022082073A2 (en) 2020-10-16 2022-04-21 Board Of Regents, The University Of Texas System Compositions and methods for muc18 targeting
WO2022081516A1 (en) 2020-10-13 2022-04-21 Janssen Biotech, Inc. Bioengineered t cell mediated immunity, materials and other methods for modulating cluster of differentiation iv &/or viii
WO2022078344A1 (en) 2020-10-12 2022-04-21 南京驯鹿医疗技术有限公司 Antibody and chimeric antigen receptor (car) binding to cd70, and application thereof
US11312785B2 (en) 2018-05-19 2022-04-26 Boehringer Ingelheim International Gmbh Antagonizing CD73 antibody
US11312783B2 (en) 2017-06-22 2022-04-26 Novartis Ag Antibody molecules to CD73 and uses thereof
WO2022083049A1 (en) 2020-10-19 2022-04-28 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
WO2022083723A1 (en) 2020-10-23 2022-04-28 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
US11326182B2 (en) 2016-04-29 2022-05-10 Voyager Therapeutics, Inc. Compositions for the treatment of disease
WO2022095803A1 (en) 2020-11-03 2022-05-12 南京北恒生物科技有限公司 Cd7-targeting humanized antibody and use thereof
WO2022096020A1 (en) 2020-11-09 2022-05-12 江苏中新医药有限公司 Anti-ecto-5'-nucleotidase antibody sequence
WO2022104009A1 (en) 2020-11-13 2022-05-19 Rubryc Therapeutics, Inc. Cd25 antibodies
WO2022105914A1 (en) 2020-11-23 2022-05-27 江苏先声药业有限公司 Antibody binding to cd70 and application thereof
WO2022105881A1 (en) 2020-11-20 2022-05-27 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Anti-cd73 antibody, antibody-drug conjugate, and use thereof
WO2022111616A1 (en) 2020-11-30 2022-06-02 石药集团巨石生物制药有限公司 Anti-cldn18.2 antibody, drug conjugate, and preparation method therefor and use thereof
WO2022125837A1 (en) 2020-12-09 2022-06-16 City Of Hope Compositions and uses of cd19 targeted chimeric antigen receptor modified immune cells
US11365258B2 (en) 2017-03-10 2022-06-21 Berlin-Chemie Ag Pharmaceutical combinations comprising an anti-LY75 antibody
WO2022127844A1 (en) 2020-12-17 2022-06-23 江苏先声药业有限公司 Cd5 antibody and use thereof
WO2022136888A1 (en) 2020-12-23 2022-06-30 Bivictrix Limited Bispecific antibodies binding to cd7 and cd33
US11377500B2 (en) 2018-02-01 2022-07-05 Pfizer Inc. Antibodies specific for CD70 and their uses
WO2022143951A1 (en) 2020-12-31 2022-07-07 康诺亚生物医药科技（成都）有限公司 Development and use of function-enhanced antibody blocking agent
WO2022147338A1 (en) 2020-12-30 2022-07-07 Ascendo Biotechnology, Inc. Monoclonal antibody against human mac-1 and uses thereof
US11384153B2 (en) 2018-07-19 2022-07-12 Regeneran Pharmaceuticals, Inc. Bispecific anti-BCMA x anti-CD3 antibodies and uses thereof
CN114763383A (en) 2021-01-13 2022-07-19 博生吉医药科技(苏州)有限公司 Monoclonal antibody targeting human BCMA and application thereof
WO2022151851A1 (en) 2021-01-12 2022-07-21 上海雅科生物科技有限公司 Cd7-targeted engineered immune cell, chimeric antigen receptor, cd7 blocking molecule and use thereof
WO2022155608A2 (en) 2021-01-15 2022-07-21 The General Hospital Corporation Cd64 chimeric receptor and uses thereof
WO2022157094A2 (en) 2021-01-22 2022-07-28 Bayer Aktiengesellschaft Lrrc15 antibodies and conjugates thereof
US20220233591A1 (en) 2021-01-25 2022-07-28 Beijing Daxi Biotechnology Co., Ltd Preparation method of nk cells and use thereof in the treatment of cancer
WO2022159737A1 (en) 2021-01-22 2022-07-28 Celldex Therapeutics, Inc. Anti-kit antibodies and uses thereof
WO2022161314A1 (en) 2021-01-27 2022-08-04 信达生物制药(苏州)有限公司 Single-domain antibody against cd16a and use thereof
WO2022166802A1 (en) 2021-02-04 2022-08-11 上海交通大学 Anti-human cd271 monoclonal antibody
US11419933B2 (en) 2016-01-25 2022-08-23 Amgen Inc. Pharmaceutical composition comprising bispecific antibody constructs
WO2022175255A2 (en) 2021-02-16 2022-08-25 Janssen Pharmaceutica Nv Trispecific antibody targeting bcma, gprc5d, and cd3
WO2022174813A1 (en) 2021-02-19 2022-08-25 信达生物制药(苏州)有限公司 Anti-gprc5d×bcma×cd3 trispecific antibody and use thereof
US11427648B2 (en) 2017-10-06 2022-08-30 Universite D'aix-Marseille Anti-CD146 antibodies and uses thereof
WO2022179039A1 (en) 2021-02-24 2022-09-01 苏州近岸蛋白质科技股份有限公司 Anti-human cd73 antibody and use thereof
WO2022187050A1 (en) 2021-03-01 2022-09-09 BioLegend, Inc. Anti-cd117 agents and compositions and methods for making and using the same
US11440966B2 (en) 2016-07-14 2022-09-13 Genmab A/S Multispecific antibodies against CD40 and CD137
US11447558B2 (en) 2017-01-03 2022-09-20 Hoffmann-La Roche Inc. Bispecific antigen binding molecules comprising anti-4-1BB clone 20H4.9
US11447548B2 (en) 2017-10-31 2022-09-20 Xenikos B.V. Immunotoxins, formulations thereof and their use in medicine
US11453721B2 (en) 2018-12-19 2022-09-27 Regeneran Pharmaceuticals, Inc. Bispecific anti-MUC16 x anti-CD28 antibodies and uses thereof
WO2022212848A1 (en) 2021-04-02 2022-10-06 Teneobio, Inc. Agonistic anti-il-2r antibodies and methods of use
WO2022214677A1 (en) 2021-04-09 2022-10-13 Cancer Research Technology Limited Anti-cd73 antibodies
WO2022221409A1 (en) 2021-04-14 2022-10-20 Villaris Therapeutics, Inc. Anti-cd122 antibodies and uses thereof
WO2022226317A1 (en) 2021-04-23 2022-10-27 Profoundbio Us Co. Anti-cd70 antibodies, conjugates thereof and methods of using the same
WO2022222910A1 (en) 2021-04-22 2022-10-27 南京北恒生物科技有限公司 Gprc5d-targeting antibody and use thereof
US11492402B2 (en) 2018-10-15 2022-11-08 Novartis Ag TREM2 stabilizing antibodies
US11492409B2 (en) 2018-06-01 2022-11-08 Novartis Ag Binding molecules against BCMA and uses thereof
WO2022241082A1 (en) 2021-05-14 2022-11-17 Genentech, Inc. Agonists of trem2
US11505606B2 (en) 2016-09-14 2022-11-22 Teneobio, Inc. CD3 binding antibodies
WO2022242758A1 (en) 2021-05-21 2022-11-24 百奥泰生物制药股份有限公司 Anti-cd73 antibody and use thereof
WO2022243838A1 (en) 2021-05-18 2022-11-24 Janssen Biotech, Inc. Compositions comprising a t cell redirection therapeutic and an anti-cd44 therapeutic
WO2022247804A1 (en) 2021-05-23 2022-12-01 上海祥耀生物科技有限责任公司 Anti-gprc5d antibody, preparation method therefor, and use thereof
WO2022247756A1 (en) 2021-05-23 2022-12-01 上海邦耀生物科技有限公司 Chimeric antigen receptor targeting gprc5d, and use thereof
US11525006B2 (en) 2017-01-23 2022-12-13 Crage Medical Co., Limited BCMA-targeting antibody and use thereof
WO2022257835A1 (en) 2021-06-08 2022-12-15 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Cd7-based humanized chimeric antigen receptor and use thereof
US11530268B2 (en) 2018-10-09 2022-12-20 Sanofi Trispecific anti-CD38, anti-CD28, and anti-CD3 binding proteins and methods of use for treating viral infection
US11530273B2 (en) 2017-05-23 2022-12-20 Helmholtz Zentrum München—Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Anti-CD73 monoclonal antibody, encoding nucleic acids and method for producing
WO2022263855A1 (en) 2021-06-18 2022-12-22 Autolus Limited Anti-cd307e single-domain antibodies, uses thereof in car t-cell and for the treatment of diseases
WO2022262101A1 (en) 2021-06-17 2022-12-22 南京蓝盾生物科技有限公司 Anti-cd70 antibody having enhanced adcc effect and application thereof
US11535672B2 (en) 2017-02-28 2022-12-27 Affimed Gmbh Combination of an anti-CD16A antibody with a cytokine
US11535869B2 (en) 2021-04-08 2022-12-27 Sana Biotechnology, Inc. CD8-specific antibody constructs and compositions thereof
US11535678B2 (en) 2016-11-21 2022-12-27 Alligator Bioscience Ab Anti-CD137 antibodies and uses thereof
WO2023274183A1 (en) 2021-06-29 2023-01-05 江苏先声药业有限公司 Cd16 antibody and use thereof
US20230002489A1 (en) 2019-11-26 2023-01-05 Shanghai Epimab Biotherapeutics Co., Ltd. Antibodies to cd3 and bcma, and bispecific binding proteins made therefrom
WO2023278520A1 (en) 2021-06-30 2023-01-05 Board Of Regents, The University Of Texas System Polypeptides targeting cd70-positive cancers
US11548947B2 (en) 2018-06-21 2023-01-10 Regeneron Pharmaceuticals, Inc. Bispecific anti-PSMA X anti-CD28 antibodies and uses thereof
WO2023280880A1 (en) 2021-07-05 2023-01-12 Trion Research Gmbh Multispecific antibodies binding to tumor-associated antigens causing no or reduced nerve pain in the treatment of cancer
US11555077B2 (en) 2017-11-09 2023-01-17 Shanghai Hyamab Biotech Co., Ltd. 4-1BB antibody and preparation method and use thereof
WO2023004304A1 (en) 2021-07-20 2023-01-26 Inhibrx, Inc. Cd8-binding polypeptides and uses thereof
WO2023012802A1 (en) 2021-08-05 2023-02-09 Yeda Research And Development Co. Ltd. Antibodies for treating cancer
US11591401B2 (en) 2020-08-19 2023-02-28 Xencor, Inc. Anti-CD28 compositions
US20230067770A1 (en) 2020-02-28 2023-03-02 Shanghai Henlius Biotech, Inc. Anti-cd137 constructs, multispecific antibody and uses thereof
WO2023026791A1 (en) 2021-08-27 2023-03-02 株式会社免疫生物研究所 Antibody fragment specific to c-kit positive tumors
US20230075244A1 (en) 2020-02-20 2023-03-09 The Feinstein Institutes For Medical Research Agonist antibodies against endoglin and uses thereof
WO2023031403A1 (en) 2021-09-02 2023-03-09 F. Hoffmann-La Roche Ag Antibodies for the treatment of aml
WO2023037015A1 (en) 2021-09-13 2023-03-16 Universiteit Gent Aminopeptidase n-specific monoclonal antibodies and uses thereof
WO2023039612A1 (en) 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Trem2 antigen binding proteins and uses thereof
WO2023039450A2 (en) 2021-09-07 2023-03-16 Duke University Compositions and methods for treatment of retinal degeneration
US11613584B1 (en) 2021-10-13 2023-03-28 Immuneonco Biopharmaceuticals (Shanghai) Inc. Antibodies binding CD70, preparation and use thereof
WO2023047100A1 (en) 2021-09-21 2023-03-30 Quell Therapeutics Ltd Anti-trem2 chimeric antigen receptor
US11623961B2 (en) 2017-11-01 2023-04-11 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for B-cell maturation antigen
US11634500B2 (en) 2019-09-06 2023-04-25 Les Laboratoires Servier Anti-CD73 antibodies and compositions
US11643456B2 (en) 2016-07-29 2023-05-09 Obi Pharma, Inc. Human antibodies, pharmaceutical compositions and methods
WO2023078382A1 (en) 2021-11-05 2023-05-11 正大天晴药业集团股份有限公司 Antibody binding to gprc5d and use thereof
US20230144142A1 (en) 2020-02-27 2023-05-11 McSAF Inside Oncology Anti-cd56 antibody-drug conjugates and their use in therapy
WO2023078113A1 (en) 2021-11-02 2023-05-11 Epimab Biotherapeutics (Hk) Limited Anti-cd122 antibodies, anti-cd132 antibodies, and related bispecific binding proteins
US20230193292A1 (en) 2020-08-20 2023-06-22 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Bcma-binding single variable structural domain and antigen-binding molecule
WO2023125728A1 (en) 2021-12-31 2023-07-06 康源博创生物科技(北京)有限公司 Anti-gprc5d antibody and use thereof
WO2023143537A1 (en) 2022-01-29 2023-08-03 恺兴生命科技(上海)有限公司 Gprc5d antibody and use thereof
US11723925B2 (en) 2017-07-05 2023-08-15 Ucl Business Ltd ROR1 CAR T-cells
US20230272089A1 (en) 2020-08-05 2023-08-31 Synthekine, Inc Il2rg binding molecules and methods of use
US20230272090A1 (en) 2020-08-05 2023-08-31 Sandro Vivona Il2rb binding molecules and methods of use
WO2023183927A2 (en) 2022-03-24 2023-09-28 Actinium Pharmaceuticals, Inc. Humanized anti-cd45 antibodies
WO2023196445A1 (en) 2022-04-05 2023-10-12 Capstan Therapeutics, Inc. Peg-lipids and lipid nanoparticles
WO2023201316A2 (en) 2022-04-13 2023-10-19 Board Of Regents, The University Of Texas System Anti-soluble interleukin-7 receptor antibody therapy to treat autoimmune diseases
US20230331872A1 (en) 2020-08-25 2023-10-19 Cytoimmune Therapeutics, Inc. Bispecific antibody car cell immunotherapy
US11814435B2 (en) 2015-04-13 2023-11-14 Pfizer Inc. Therapeutic antibodies and their uses
WO2023235772A2 (en) 2022-05-31 2023-12-07 Fred Hutchinson Cancer Center Humanized anti-cd45 antibodies and uses thereof
WO2023236889A1 (en) 2022-06-06 2023-12-14 山东先声生物制药有限公司 Multi-specific antibody targeting bcma, gprc5d and t cells and application thereof
US20240034816A1 (en) 2022-05-27 2024-02-01 Sanofi NATURAL KILLER (NK) CELL ENGAGERS BINDING TO NKp46 AND BCMA VARIANTS WITH FC-ENGINEERING
WO2024031091A2 (en) 2022-08-05 2024-02-08 Juno Therapeutics, Inc. Chimeric antigen receptors specific for gprc5d and bcma
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
US11919965B2 (en) 2019-12-06 2024-03-05 Regeneron Pharmaceuticals, Inc. Methods of treating multiple myeloma with bispecific anti-BCMA x anti-CD3 antibodies
WO2024047558A2 (en) 2022-08-31 2024-03-07 Immuneel Therapeutics Private Limited Antigen-binding proteins and chimeric antigen receptors specific for domain 9 of human cd307e
WO2024046239A1 (en) 2022-08-30 2024-03-07 苏州缔码生物科技有限公司 Recombinant humanized monoclonal antibody targeting human gprc5d and application thereof
WO2024064771A1 (en) 2022-09-20 2024-03-28 Vor Biopharma Inc. Anti-cd45-ign antibody drug conjugates and uses thereof
WO2024073723A2 (en) 2022-09-30 2024-04-04 Forte Subsidiary, Inc. Anti-cd122 antibodies and uses thereof
WO2024079015A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and imids
WO2024081729A2 (en) 2022-10-12 2024-04-18 Mythic Therapeutics, Inc. Lrrc-15-binding protein constructs and uses thereof
WO2024086190A1 (en) 2022-10-18 2024-04-25 Kite Pharma, Inc. Novel cd19 binders, car-t constructs comprising the same, and methods of using the same
WO2024102770A1 (en) 2022-11-07 2024-05-16 Tessera Therapeutics, Inc. Lipid nanoparticle drug conjugates
WO2024131962A1 (en) 2022-12-23 2024-06-27 成都恩沐生物科技有限公司 Novel anti-gprc5d antibody
US20240228610A9 (en) 2022-10-10 2024-07-11 Medimmune, Llc Compositions and methods of treating cancer with chimeric antigen receptors targeting claudin 18.2
WO2024158047A1 (en) 2023-01-27 2024-08-02 第一三共株式会社 Anti-lrrc15 antibody
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL315577A (en) * 2022-04-05 2024-11-01 Capstan Therapeutics Inc Ionized cationic lipids and lipid nanoparticles
WO2025076113A1 (en) * 2023-10-05 2025-04-10 Capstan Therapeutics, Inc. Ionizable cationic lipids with conserved spacing and lipid nanoparticles
WO2025129201A1 (en) * 2023-12-15 2025-06-19 Capstan Therapeutics, Inc. Humanized anti-cd8 antibodies and uses thereof

Patent Citations (443)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989006968A1 (en) 1988-02-03 1989-08-10 Xoma Corporation Therapeutic use of anti-t cell immunotoxin for autoimmune diseases
US5283173A (en) 1990-01-24 1994-02-01 The Research Foundation Of State University Of New York System to detect protein-protein interactions
US5468614A (en) 1990-01-24 1995-11-21 The Research Foundation Of State University Of New York System to detect protein-protein interactions
WO2001023573A1 (en) 1999-09-30 2001-04-05 Kyowa Hakko Kogyo Co., Ltd. Human type complementation-determining domain transplanted antibody against ganglioside gd2 and derivative of this antibody
WO2002030459A1 (en) 2000-10-11 2002-04-18 Tegenero Gmbh Use of cd28-specific monoclonal antibodies for stimulating blood cells that lack cd28
WO2002047721A1 (en) 2000-12-14 2002-06-20 Fujisawa Pharmaceutical Co., Ltd. Silensed anti-cd28 antibodies and use thereof
US7825222B2 (en) 2001-02-12 2010-11-02 Novartis Ag Therapeutic binding molecules
US6924360B2 (en) 2001-12-28 2005-08-02 Abgenix, Inc. Antibodies against the MUC18 antigen
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
WO2004045512A2 (en) 2002-11-15 2004-06-03 Genmab A/S Human monoclonal antibodies against cd25
WO2004055056A1 (en) 2002-12-17 2004-07-01 Merck Patent Gmbh Humanized antibody (h14.18) of the mouse 14.18 antibody binding to gd2 and its fusion with il-2
WO2005035584A1 (en) 2003-10-10 2005-04-21 Bristol-Myers Squibb Company Fully human antibodies against human 4-1bb (cd137)
WO2005037999A2 (en) 2003-10-14 2005-04-28 Biogen Idec Ma Inc. Treatment of cancer using antibodies to lrrc15
US20100040540A1 (en) 2003-11-19 2010-02-18 Institut National De La Sante Et De La Recherche Medicale (Inserm) Anti cd44 antibodies for eradicating leukaemic stem cells and breast cancer stem cells
US9617349B2 (en) 2004-01-22 2017-04-11 Merck Patent Gmbh Anti-cancer antibodies with reduced complement fixation
US7378504B2 (en) 2004-06-03 2008-05-27 Medarex, Inc. Human monoclonal antibodies to Fc gamma receptor I (CD64)
US8383117B2 (en) 2004-06-10 2013-02-26 Viventia Biotechnologies Inc. CD44E tumor specific antibody
US10385137B2 (en) 2004-12-16 2019-08-20 Inserm (Institut National De La Sante Et De La Recherche Medicale) Production of antibody formats and immunological applications of said formats
US9284375B2 (en) 2005-04-15 2016-03-15 Macrogenics, Inc. Covalent diabodies and uses thereof
WO2006108670A2 (en) 2005-04-15 2006-10-19 Novartis Ag Use of cd25 antibodies in immunotherapy
US9701752B2 (en) 2005-04-19 2017-07-11 Seattle Genetics, Inc. Humanized anti-CD70 binding agents and uses thereof
WO2007009065A2 (en) 2005-07-11 2007-01-18 Macrogenics, Inc. Methods of treating autoimmune disease using humanized anti-cd16a antibodies
US8278036B2 (en) 2005-08-23 2012-10-02 The Trustees Of The University Of Pennsylvania RNA containing modified nucleosides and methods of use thereof
US8124738B2 (en) 2005-09-26 2012-02-28 Medarex, Inc. Human monoclonal antibodies to CD70
WO2007084321A2 (en) 2006-01-12 2007-07-26 Alexion Pharmaceuticals, Inc. Antibodies to ox-2/cd200 and uses thereof
US7915391B2 (en) 2006-04-24 2011-03-29 Amgen Inc. Humanized c-Kit antibody
US9388249B2 (en) 2006-07-10 2016-07-12 Amano Enzyme Inc. Method of classifying antibody, method of identifying antigen, method of obtaining antibody or antibody set, method of constructing antibody panel and antibody or antibody set and use of the same
US8951524B2 (en) 2006-10-10 2015-02-10 Universite De Nantes Use of monoclonal antibodies specific to the O-acetylated form of GD2 ganglioside for treatment of certain cancers
WO2008121160A2 (en) 2006-11-21 2008-10-09 Xencor, Inc. Optimized antibodies that target cd5
WO2008079246A2 (en) 2006-12-21 2008-07-03 Medarex, Inc. Cd44 antibodies
WO2008117049A1 (en) 2007-03-26 2008-10-02 Affitech As Human anti cd166 antibody binding human tumor cells
US7722869B2 (en) 2007-04-27 2010-05-25 Novartis Ag Antibody molecules and nucleic acids
US9688755B2 (en) 2007-05-14 2017-06-27 Novimmune S.A. Fc receptor-binding polypeptides with modified effector functions
WO2008144890A1 (en) 2007-05-30 2008-12-04 F. Hoffmann-La Roche Ag Chimeric and humanized anti-cd44 antibodies that mediate cancer cell cytotoxicity
US8722858B2 (en) 2007-06-25 2014-05-13 Chugai Seiyaku Kabushiki Kaisha Anti-Prominin-1 antibody having ADCC activity or CDC activity
US8236318B2 (en) 2007-11-07 2012-08-07 Celldex Therapeutics Inc. Antibodies that bind human dendritic and epithelial cell 205 (DEC-205)
US20170335016A1 (en) 2008-04-25 2017-11-23 Kyowa Hakko Kirin Co., Ltd Stable multivalent antibody
US8507657B2 (en) 2008-06-30 2013-08-13 Morphotek, Inc. Anti-GD2 antibodies
US8454959B2 (en) 2008-07-18 2013-06-04 Bristol-Meyers Squibb Company Compositions monovalent for CD28 binding and methods of use
WO2010022737A1 (en) 2008-08-29 2010-03-04 Symphogen A/S Anti-cd5 antibodies
WO2010032059A2 (en) 2008-09-19 2010-03-25 Medimmune Llc Targeted binding agents directed to cd105 and uses thereof
WO2010039873A2 (en) 2008-10-01 2010-04-08 Tracon Pharmaceuticals, Inc. Humanized endoglin antibodies
WO2010104949A2 (en) 2009-03-10 2010-09-16 Biogen Idec Ma Inc. Anti-bcma antibodies
US8679500B2 (en) 2009-05-14 2014-03-25 Institute National De La Sante Et De La Recherche Medicale (Inserm) Compositions containing antibodies for treating CD5+ HLA-DR+ B or T cell related diseases
US8221753B2 (en) 2009-09-30 2012-07-17 Tracon Pharmaceuticals, Inc. Endoglin antibodies
WO2011094259A2 (en) 2010-01-28 2011-08-04 Glaxo Group Limited Cd127 binding proteins
US8785604B2 (en) 2010-02-18 2014-07-22 Effimune Anti-CD28 humanized antibodies
WO2011104687A1 (en) 2010-02-24 2011-09-01 Rinat Neuroscience Corporation Antagonist anti-il-7 receptor antibodies and methods
WO2011127324A2 (en) 2010-04-08 2011-10-13 JN Biosciences, LLC Antibodies to cd122
US20110280881A1 (en) 2010-04-13 2011-11-17 Riken Novel anti-hsp90 monoclonal antibody
US9249225B2 (en) 2010-05-26 2016-02-02 Regents Of The University Of Minnesota Single chain variable fragment anti-CD133 antibodies and uses thereof
US9315585B2 (en) 2010-06-19 2016-04-19 Memorial Sloan Kettering Cancer Center Anti-GD2 antibodies
US9220772B2 (en) 2010-07-22 2015-12-29 The Regents Of The University Of California Anti-tumor antigen antibodies and methods of use
US9493740B2 (en) 2010-09-08 2016-11-15 Baylor College Of Medicine Immunotherapy of cancer using genetically engineered GD2-specific T cells
WO2012032433A1 (en) 2010-09-09 2012-03-15 Pfizer Inc. 4-1bb binding molecules
US9115192B2 (en) 2010-09-27 2015-08-25 Zestagen S.A. Compositions and methods for treating neoplasia
US10273294B2 (en) 2010-10-11 2019-04-30 University Of Southern California Compositions and methods for treating HIF-1α over-expressing cancers
WO2012071216A2 (en) 2010-11-22 2012-05-31 Duke University Antibodies for tumor gangliosides
US9328156B2 (en) 2010-12-09 2016-05-03 The Trustees Of The University Of Pennsylvania Use of chimeric antigen receptor-modified T cells to treat cancer
US9540443B2 (en) 2011-01-26 2017-01-10 Kolltan Pharmaceuticals, Inc. Anti-kit antibodies
US9765148B2 (en) 2011-03-16 2017-09-19 Argen-X N.V. Antibodies to CD70
WO2012138537A1 (en) 2011-04-01 2012-10-11 Immunogen, Inc. Compositions and methods for treatment of ovarian, peritoneal, and fallopian tube cancer
WO2012149412A2 (en) 2011-04-29 2012-11-01 Immunogen Inc. Anti-endoglin (cd105) antibodies, immunoconjugates, and uses thereof
US8552157B2 (en) 2011-05-12 2013-10-08 Imclone Llc c-Kit antibodies and uses thereof
US9273141B2 (en) 2011-05-27 2016-03-01 Glaxo Group Limited B cell maturation antigen (BCMA) binding proteins
WO2012163805A1 (en) 2011-05-27 2012-12-06 Glaxo Group Limited Bcma (cd269/tnfrsf17) -binding proteins
US10266604B2 (en) 2011-09-22 2019-04-23 Amgen Inc. CD27L antigen binding proteins
WO2013056984A1 (en) 2011-10-19 2013-04-25 Effimune Antibodies directed against the alpha chain of il7 receptor - their use for the preparation of drug candidates
US20140308301A1 (en) 2011-10-26 2014-10-16 The Regents Of The University Of California Cd44 monoclonal antibody for the treatment of b-cell chronic lymphocytic leukemia and other hematological malignancies
US20130115274A1 (en) 2011-11-04 2013-05-09 Nitto Denko Corporation Method of producing lipid nanoparticles for drug delivery
WO2013167259A1 (en) 2012-05-09 2013-11-14 Ganymed Pharmaceuticals Ag Antibodies against claudin 18.2 useful in cancer diagnosis
US9777068B2 (en) 2012-06-18 2017-10-03 Apeiron Biologics Ag Method for treating a GD2 positive cancer
WO2014000338A1 (en) 2012-06-28 2014-01-03 中国科学院生物物理研究所 Application of composite of anti-cd146 antibody and anti-vegf antibody in preparing medicine for suppressing tumor
US10414825B2 (en) 2012-09-10 2019-09-17 Prothena Biosciences Limited Anti-MCAM antibodies and associated methods of use
US9447190B2 (en) 2012-09-10 2016-09-20 Prothena Biosciences Limited Anti-MCAM antibodies and associated methods of use
WO2014083379A1 (en) 2012-11-30 2014-06-05 Institut Pasteur Use of anti-fcyri and/or anti-fcyriia antibodies for treating arthritis, inflammation, thrombocytopenia and allergic shock
US9243058B2 (en) 2012-12-07 2016-01-26 Amgen, Inc. BCMA antigen binding proteins
WO2014128185A1 (en) 2013-02-21 2014-08-28 Universitätsklinikum Freiburg Recombinant bispecific antibody that binds to the cd133 antigen on tumor cells and to the human cd3 t cell receptor
WO2014158821A1 (en) 2013-03-12 2014-10-02 Imaginab, Inc. Antigen binding constructs to cd70
US10414820B2 (en) 2013-03-13 2019-09-17 Imaginab, Inc. Antigen binding constructs to CD8
US9789203B2 (en) 2013-03-15 2017-10-17 Novartis Ag cKIT antibody drug conjugates
US9782500B2 (en) 2013-09-24 2017-10-10 Oncoinvent As Monoclonal antibody and derivatives
WO2015052538A1 (en) 2013-10-10 2015-04-16 Ucl Business Plc Chimeric antigen receptor
US10081682B2 (en) 2013-10-11 2018-09-25 Oxford Bio Therapeutics Ltd. Conjugated antibodies against LY75 for the treatment of cancer
US9932410B2 (en) 2013-11-05 2018-04-03 Inserm (Institut National De La Sante Et De La Recherche Medicale Human neutralizing anti-kit antibodies and uses thereof
WO2015076425A1 (en) 2013-11-25 2015-05-28 リンク・ジェノミクス株式会社 New monoclonal antibody
WO2015118031A2 (en) 2014-02-06 2015-08-13 Oncomatryx Biopharma, S.L. Antibody-drug conjugates and immunotoxins
US10106623B2 (en) 2014-02-12 2018-10-23 Michael Uhlin Bispecific antibodies for use in stem cell transplantation
WO2015132604A1 (en) 2014-03-06 2015-09-11 Ucl Business Plc Chimeric antigen receptor
US10407506B2 (en) 2014-03-12 2019-09-10 Prothena Biosciences Limited Anti-MCAM antibodies and associated methods of use
WO2015140591A1 (en) 2014-03-21 2015-09-24 Nordlandssykehuset Hf Anti-cd14 antibodies and uses thereof
US20170166638A1 (en) 2014-03-26 2017-06-15 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. USE OF CD-64-BLOCKING AGENTS AS ENHANCER OF AN ANTI-TNF-a ANTIBODY THERAPY
US10316101B2 (en) 2014-04-14 2019-06-11 Cellectis BCMA (CD269) specific chimeric antigen receptors for cancer immunotherapy
US20150337039A1 (en) 2014-05-22 2015-11-26 Case Western Reserve University Mac-1 antibodies and uses thereof
WO2015184203A1 (en) 2014-05-29 2015-12-03 Macrogenics, Inc. Tri-specific binding molecules and methods of use thereof
WO2015187528A1 (en) 2014-06-02 2015-12-10 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting cd-19
US10287350B2 (en) 2014-06-02 2019-05-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Chimeric antigen receptors targeting CD-19
US10106609B2 (en) 2014-06-04 2018-10-23 PeronGen BioTherapeutics (Suzhou) Co., Ltd. CD7 nanobodies, encoding sequence and use thereof
US9856324B2 (en) 2014-06-04 2018-01-02 MabVax Therapeutics, Inc. Human monoclonal antibodies to ganglioside GD2
WO2015189302A1 (en) 2014-06-10 2015-12-17 Effimune Antibodies directed against cd127
US10752684B2 (en) 2014-07-29 2020-08-25 Cellectis ROR1 (NTRKR1) specific chimeric antigen receptors for cancer immunotherapy
US10273295B2 (en) 2014-08-19 2019-04-30 Miltenyi Biotec Gmbh Chimeric antigen receptor specific for SSEA4 antigen
US10406179B2 (en) 2014-08-26 2019-09-10 The Board Of Trustees Of The Leland Stanford Junior University Engraftment of stem cells with a combination of an agent that targets stem cells and modulation of immunoregulatory signaling
US9938356B2 (en) 2014-11-10 2018-04-10 Medimmune Limited Binding molecules specific for CD73 and uses thereof
US9926375B2 (en) 2014-11-12 2018-03-27 Tracon Pharmaceuticals, Inc. Anti-endoglin antibodies and uses thereof
US10100129B2 (en) 2014-11-21 2018-10-16 Bristol-Myers Squibb Company Antibodies against CD73 and uses thereof
US9382321B2 (en) 2014-11-26 2016-07-05 Adventis Health System/Sunbelt, Inc. Effector-deficient anti-CD32A antibodies
US9701756B2 (en) 2014-11-26 2017-07-11 Miltenyi Biotec Gmbh Humanized antibody or fragment thereof specific for CD45R0
WO2016090337A1 (en) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Chimeric antigen receptors targeting fc receptor-like 5 and uses thereof
WO2016090329A2 (en) 2014-12-05 2016-06-09 Memorial Sloan-Kettering Cancer Center Antibodies targeting g-protein coupled receptor and methods of use
US10689456B2 (en) 2014-12-08 2020-06-23 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-CD70 chimeric antigen receptors
US10383929B2 (en) 2014-12-12 2019-08-20 Bluebird Bio, Inc. BCMA chimeric antigen receptors
WO2016130598A1 (en) 2015-02-09 2016-08-18 University Of Florida Research Foundation, Inc. Bi-specific chimeric antigen receptor and uses thereof
US11248058B2 (en) 2015-02-19 2022-02-15 University Of Florida Research Foundation, Inc. Chimeric antigen receptors and uses thereof
US10738121B2 (en) 2015-02-27 2020-08-11 The General Hospital Corporation Therapeutic use of integrin-binding antibodies
WO2016149578A1 (en) 2015-03-19 2016-09-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Dual specific anti-cd22-anti-cd19 chimeric antigen receptors
US10711068B2 (en) 2015-03-26 2020-07-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-CD133 monoclonal antibodies and related compositions and methods
WO2016166630A1 (en) 2015-04-13 2016-10-20 Pfizer Inc. Chimeric antigen receptors targeting b-cell maturation antigen
US11814435B2 (en) 2015-04-13 2023-11-14 Pfizer Inc. Therapeutic antibodies and their uses
US10758625B2 (en) 2015-04-14 2020-09-01 Benhealth Biopharmaceutic (Shenzhen) Co., Ltd. Bispecific antibody capable of being combined with immune cells to enhance tumor killing capability, and preparation method therefor and application thereof
US10745481B2 (en) 2015-05-04 2020-08-18 Cytomx Therapeutics, Inc. Anti-CD166 antibodies, activatable anti-CD166 antibodies, and methods of use thereof
WO2016187216A1 (en) 2015-05-18 2016-11-24 Bluebird Bio, Inc. Anti-ror1 chimeric antigen receptors
US10920246B2 (en) 2015-05-26 2021-02-16 Ramot At Tel-Aviv University Ltd. Targeted lipid particles for systemic delivery of nucleic acid molecules to leukocytes
WO2016197974A1 (en) 2015-06-12 2016-12-15 Yen-Ta Lu Methods and antibodies for modulation of immunoresponse
WO2017009473A1 (en) 2015-07-16 2017-01-19 Ucb Biopharma Sprl Antibody molecules which bind cd45
US10730941B2 (en) 2015-07-31 2020-08-04 Memorial Sloan-Kettering Cancer Center Antigen-binding proteins targeting CD56 and uses thereof
US10548987B2 (en) 2015-07-31 2020-02-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Antibody-drug conjugates for targeting CD56-positive tumors
US10683369B2 (en) 2015-08-03 2020-06-16 Engmab Sàrl Monoclonal antibodies against BCMA
WO2017021540A1 (en) 2015-08-06 2017-02-09 Agency For Science, Technology And Research Il2rbeta/common gamma chain antibodies
US10934363B2 (en) 2015-08-11 2021-03-02 Legend Biotech Usa Inc. Chimeric antigen receptors based on single domain antibodies and methods of use thereof
WO2018028647A1 (en) 2015-08-11 2018-02-15 Legend Biotech Usa Inc. Chimeric antigen receptors targeting bcma and methods of use thereof
US20170051068A1 (en) 2015-08-17 2017-02-23 Janssen Pharmaceutica Nv Anti-BCMA Antibodies, Bispecific Antigen Binding Molecules that Bind BCMA and CD3, and Uses Thereof
US10072088B2 (en) 2015-08-17 2018-09-11 Janssen Pharmaceutica, Nv Anti-BCMA antibodies and uses thereof
WO2017048770A1 (en) 2015-09-15 2017-03-23 Regulus Therapeutics, Inc. Systems, compositions, and methods for formulating nucleic acid compositions
WO2017058866A1 (en) 2015-09-28 2017-04-06 Precision Immune, Inc. Anti-trem2 antibodies and uses thereof
WO2017055385A1 (en) 2015-10-02 2017-04-06 F. Hoffmann-La Roche Ag Anti-cd3xgd2 bispecific t cell activating antigen binding molecules
WO2017062748A1 (en) 2015-10-07 2017-04-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Il-7r-alpha specific antibodies for treating acute lymphoblastic leukemia
WO2017064221A1 (en) 2015-10-13 2017-04-20 Affimed Gmbh Multivalent fv antibodies
WO2017096120A1 (en) 2015-12-04 2017-06-08 Memorial Sloan-Kettering Cancer Center Antibodies targeting fc receptor-like 5 and methods of use
US10822426B2 (en) 2015-12-09 2020-11-03 Corvus Pharmaceuticals, Inc. Humanized anti-CD73 antibodies
WO2017118613A1 (en) 2016-01-08 2017-07-13 Syddansk Universitet Bispecific antibodies targeting human cd73
WO2017123650A2 (en) 2016-01-11 2017-07-20 Inhibrx Lp Multivalent and multispecific 41bb-binding fusion proteins
US11419933B2 (en) 2016-01-25 2022-08-23 Amgen Inc. Pharmaceutical composition comprising bispecific antibody constructs
WO2017130223A2 (en) 2016-01-29 2017-08-03 Virocan Therapeutics Pvt. Ltd. A chimeric antigen receptor specific to b-cell maturation antigen, a recombinant expression vector and a method thereof
WO2017134306A1 (en) 2016-02-05 2017-08-10 Orionis Biosciences Nv Cd8 binding agents
US11078291B2 (en) 2016-02-17 2021-08-03 Seagen Inc. BCMA antibodies and use of same to treat cancer and immunological disorders
WO2017156479A1 (en) 2016-03-11 2017-09-14 Bluebird Bio, Inc. Ror1 chimeric antigen receptors
WO2017173256A1 (en) 2016-04-01 2017-10-05 Kite Pharma, Inc. Chimeric antigen and t cell receptors and methods of use
US10633444B2 (en) 2016-04-18 2020-04-28 Celldex Therapeutics, Inc. Agonistic antibodies that bind CD40
US11326182B2 (en) 2016-04-29 2022-05-10 Voyager Therapeutics, Inc. Compositions for the treatment of disease
US11306145B2 (en) 2016-04-29 2022-04-19 Viela Bio, Inc. Binding molecules specific for FcγRIIA and uses thereof
WO2017213979A1 (en) 2016-06-06 2017-12-14 St. Jude Children's Research Hospital Anti-cd7 chimeric antigen receptor and method of use thereof
US10919967B2 (en) 2016-06-21 2021-02-16 Albert-Ludwigs-Universitätfreiburg Antibodies against Mac-1
US10457726B2 (en) 2016-06-30 2019-10-29 University Of Connecticut Antibody and antigen-binding fragment compositions targeting cell surface antigens in tumors and methods of use thereof
WO2018006882A1 (en) 2016-07-08 2018-01-11 科济生物医药（上海）有限公司 Antibody for anti-claudin 18a2 and use thereof
US11440966B2 (en) 2016-07-14 2022-09-13 Genmab A/S Multispecific antibodies against CD40 and CD137
WO2018010846A1 (en) 2016-07-15 2018-01-18 Ogd2 Pharma Humanized antibody against o-acetylated gd2 ganglioside (oacgd2)
WO2018017786A2 (en) 2016-07-20 2018-01-25 Janssen Pharmaceutica Nv Anti- gprc5d antibodies, bispecific antigen binding molecules that bind gprc5d and cd3, and uses thereof
US11643456B2 (en) 2016-07-29 2023-05-09 Obi Pharma, Inc. Human antibodies, pharmaceutical compositions and methods
WO2018039626A1 (en) 2016-08-25 2018-03-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Human single domain antibodies targeting cd16a to mediate adcc
WO2018045880A1 (en) 2016-09-06 2018-03-15 Guangzhou Bainifu Biotech Co., Ltd Chimeric antigen receptor, car133-nkt cells and use thereof
US11505606B2 (en) 2016-09-14 2022-11-22 Teneobio, Inc. CD3 binding antibodies
US10108123B2 (en) 2016-09-29 2018-10-23 Canon Kabushiki Kaisha Image forming apparatus
WO2018072025A1 (en) 2016-10-19 2018-04-26 The Governing Council Of The University Of Toronto Cd133-binding agents and uses thereof
US11535678B2 (en) 2016-11-21 2022-12-27 Alligator Bioscience Ab Anti-CD137 antibodies and uses thereof
WO2018098306A1 (en) 2016-11-22 2018-05-31 National University Of Singapore Blockade of cd7 expression and chimeric antigen receptors for immunotherapy of t-cell malignancies
US11180554B2 (en) 2016-12-13 2021-11-23 Astellas Pharma Inc. Anti-human CD73 antibody
US20190321404A1 (en) 2016-12-30 2019-10-24 Nanjing Legend Biotech Co., Ltd. Novel chimeric antigen receptor and use thereof
US11447558B2 (en) 2017-01-03 2022-09-20 Hoffmann-La Roche Inc. Bispecific antigen binding molecules comprising anti-4-1BB clone 20H4.9
US10174122B2 (en) 2017-01-06 2019-01-08 Eutilex Co., Ltd. Anti-human 4-1BB antibodies and uses thereof
US11525006B2 (en) 2017-01-23 2022-12-13 Crage Medical Co., Limited BCMA-targeting antibody and use thereof
US10584169B2 (en) 2017-01-24 2020-03-10 I-Mab Biopharma Us Limited Anti-CD73 antibodies and uses thereof
WO2018147245A1 (en) 2017-02-07 2018-08-16 第一三共株式会社 Anti-gprc5d antibody and molecule containing same
US11046775B2 (en) 2017-02-14 2021-06-29 Kite Pharma, Inc. CD70 binding molecules and methods of use thereof
WO2018152516A1 (en) 2017-02-20 2018-08-23 Adimab, Llc Proteins binding cd33, nkg2d and cd16
US20200181260A1 (en) 2017-02-22 2020-06-11 H. Lee Moffitt Cancer Center And Research Institute, Inc. Bispecific antibody for cancer immunotherapy
WO2018158349A1 (en) 2017-02-28 2018-09-07 Affimed Gmbh Tandem diabody for cd16a-directed nk-cell engagement
US11535672B2 (en) 2017-02-28 2022-12-27 Affimed Gmbh Combination of an anti-CD16A antibody with a cytokine
US11365258B2 (en) 2017-03-10 2022-06-21 Berlin-Chemie Ag Pharmaceutical combinations comprising an anti-LY75 antibody
US10426797B2 (en) 2017-03-24 2019-10-01 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-CD33 immunotherapy
US11242385B2 (en) 2017-04-13 2022-02-08 Agenus Inc. Anti-CD137 antibodies and methods of use thereof
WO2018191786A1 (en) 2017-04-21 2018-10-25 Implicit Bioscience Pty Ltd Cd 14 antagonist antibodies for treating neurodegenerative diseases
US11186636B2 (en) 2017-04-21 2021-11-30 Amgen Inc. Anti-human TREM2 antibodies and uses thereof
US20200190165A1 (en) 2017-04-28 2020-06-18 Ajinomoto Co., Inc. Compound having affinity substance to soluble protein, cleavable portion and reactive group, or salt thereof
WO2018197675A1 (en) 2017-04-28 2018-11-01 Julius-Maximilians-Universität Würzburg Ror1-specific chimeric antigen receptors (car) with humanized targeting domains
WO2018199337A1 (en) 2017-04-28 2018-11-01 味の素株式会社 Compound having substance that has affinity for soluble protein, cleavable moiety, and reactive group, or salt thereof
WO2018213337A1 (en) 2017-05-15 2018-11-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Bicistronic chimeric antigen receptors and their uses
US11530273B2 (en) 2017-05-23 2022-12-20 Helmholtz Zentrum München—Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Anti-CD73 monoclonal antibody, encoding nucleic acids and method for producing
WO2018220467A1 (en) 2017-05-30 2018-12-06 Foroogh Nejatollahi Anti-muc18 human immunotoxin and applications thereof
US11312783B2 (en) 2017-06-22 2022-04-26 Novartis Ag Antibody molecules to CD73 and uses thereof
WO2018235247A1 (en) 2017-06-22 2018-12-27 学校法人順天堂 ANTI-CD10 ANTIBODIES
WO2019006072A1 (en) 2017-06-30 2019-01-03 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-b-cell maturation antigen chimeric antigen receptors with human domains
US11723925B2 (en) 2017-07-05 2023-08-15 Ucl Business Ltd ROR1 CAR T-cells
US10716851B2 (en) 2017-07-11 2020-07-21 Compass Therapeutics Llc Agonist antibodies that bind human CD137 and uses thereof
US11220544B2 (en) 2017-07-14 2022-01-11 Cytomx Therapeutics, Inc. Anti-CD166 antibodies and uses thereof
US10730944B2 (en) 2017-07-24 2020-08-04 Regeneron Pharmaceuticals, Inc. Anti-CD8 antibodies and uses thereof
US10442867B2 (en) 2017-07-31 2019-10-15 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-CD19/CD20 immunotherapy
US10988546B2 (en) 2017-08-01 2021-04-27 Medimmune, Llc BCMA monoclonal antibody-drug conjugate
US10676525B2 (en) 2017-08-03 2020-06-09 Alector Llc Anti-TREM2 antibodies and methods of use thereof
WO2019032661A1 (en) 2017-08-09 2019-02-14 Orionis Biosciences Inc. Cd8 binding agents
WO2019033043A2 (en) 2017-08-11 2019-02-14 Genentech, Inc. Anti-cd8 antibodies and uses thereof
US11242395B2 (en) 2017-08-21 2022-02-08 Adagene Inc. Anti-CD137 molecules and use thereof
WO2019059771A2 (en) 2017-09-21 2019-03-28 Umc Utrecht Holding B.V. Anti-gd2 antibody for the treatment of neuroblastoma
US11427648B2 (en) 2017-10-06 2022-08-30 Universite D'aix-Marseille Anti-CD146 antibodies and uses thereof
US11136403B2 (en) 2017-10-13 2021-10-05 Harpoon Therapeutics, Inc. Trispecific proteins and methods of use
US10543263B2 (en) 2017-10-16 2020-01-28 Lentigen Technology Inc. Compositions and methods for treating cancer with anti-CD22 immunotherapy
WO2019084067A1 (en) 2017-10-24 2019-05-02 Magenta Therapeutics, Inc. Anti-cd117 antibodies and methods of using same
US11447548B2 (en) 2017-10-31 2022-09-20 Xenikos B.V. Immunotoxins, formulations thereof and their use in medicine
US11623961B2 (en) 2017-11-01 2023-04-11 Juno Therapeutics, Inc. Antibodies and chimeric antigen receptors specific for B-cell maturation antigen
US11292846B2 (en) 2017-11-02 2022-04-05 Bayer Aktiengesellschaft Bispecific antibodies binding ALK-1 and BMPR-2
WO2019085102A1 (en) 2017-11-03 2019-05-09 西安宇繁生物科技有限责任公司 Bcma-specific chimeric antigen receptor t cell and application thereof
US10428141B2 (en) 2017-11-03 2019-10-01 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-ROR1 immunotherapy
US11555077B2 (en) 2017-11-09 2023-01-17 Shanghai Hyamab Biotech Co., Ltd. 4-1BB antibody and preparation method and use thereof
WO2019108863A1 (en) 2017-11-29 2019-06-06 Magenta Therapeutics, Inc. Compositions and methods for the depletion of cd5+ cells
US10508148B2 (en) 2017-12-12 2019-12-17 Pionyr Immunotherapeutics, Inc. Anti-TREM2 antibodies and related methods
WO2019126536A1 (en) 2017-12-20 2019-06-27 Alexion Pharmaceuticals Inc. Humanized anti-cd200 antibodies and uses thereof
WO2019119822A1 (en) 2017-12-23 2019-06-27 Uwell Biopharma Inc. Pharmaceutical chimeric receptor composition and method thereof
WO2019133639A2 (en) 2017-12-26 2019-07-04 The Regents Of The University Of California Human antibodies that bind and are internalized by mesothelioma and other cancer cells
WO2019137309A1 (en) 2018-01-09 2019-07-18 南京任诺药业有限公司 Humanized anti-human cd146 monoclonal antibody having high neutralizing activity
US11026975B2 (en) 2018-02-01 2021-06-08 Nanjing Iaso Biotherapeutics Co., Ltd. Chimeric antigen receptor (CAR) binding to BCMA, and uses thereof
US20200246381A1 (en) 2018-02-01 2020-08-06 Nanjing Iaso Biotherapeutics Co., Ltd. Chimeric antigen receptor (car) binding to bcma, and uses thereof
US20200339699A1 (en) 2018-02-01 2020-10-29 Innovent Biologics (Suzhou) Co., Ltd. Fully humanized anti-b cell maturation antigen (bcma) single-chain antibody and use thereof
US11377500B2 (en) 2018-02-01 2022-07-05 Pfizer Inc. Antibodies specific for CD70 and their uses
WO2019154890A1 (en) 2018-02-09 2019-08-15 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
US10611838B2 (en) 2018-02-09 2020-04-07 Ultrahuman Five Limited C-KIT antibodies
WO2019159193A1 (en) 2018-02-13 2019-08-22 Indian Institute Of Technology Bombay Novel humanized anti-cd19 chimeric antigen receptor, its nucelic acid sequence and its preparation
US20210002384A1 (en) 2018-02-15 2021-01-07 St. Jude Children's Research Hospital Process for producing hu14.18k322a monoclonal antibody
WO2019170131A1 (en) 2018-03-07 2019-09-12 复旦大学 Targeted cd73 antibody and antibody-drug conjugate, and preparation method therefor and uses thereof
US11299550B2 (en) 2018-03-09 2022-04-12 Phanes Therapeutics, Inc. Anti-CD73 antibodies and uses thereof
WO2019173692A2 (en) 2018-03-09 2019-09-12 Agenus Inc. Anti-cd73 antibodies and methods of use thereof
WO2019175215A1 (en) 2018-03-13 2019-09-19 Tusk Therapeutics Ltd Anti-cd25 for tumour specific cell depletion
WO2019175223A1 (en) 2018-03-13 2019-09-19 Tusk Therapeutics Ltd Anti-cd25 for tumour specific cell depletion
WO2019175226A1 (en) 2018-03-13 2019-09-19 Tusk Therapeutics Ltd Anti-cd25 antibody agents
US20210130483A1 (en) 2018-03-26 2021-05-06 Sutro Biopharma, Inc. Anti-bcma receptor antibodies, compositions comprising anti bcma receptor antibodies and methods of making and using anti-bcma antibodies
WO2019190952A1 (en) 2018-03-29 2019-10-03 Lan Bo Chen Monoclonal antibodies that bind to ssea4 and uses thereof
US11446379B2 (en) 2018-03-29 2022-09-20 Cho Pharma Usa, Inc. Monoclonal antibodies that bind to SSEA4 and uses thereof
WO2019219089A1 (en) 2018-05-18 2019-11-21 Bridge Health Bio-Tech Co., Ltd Anti-claudin 18.2 antibodies and uses thereof
US11312785B2 (en) 2018-05-19 2022-04-26 Boehringer Ingelheim International Gmbh Antagonizing CD73 antibody
US11492409B2 (en) 2018-06-01 2022-11-08 Novartis Ag Binding molecules against BCMA and uses thereof
WO2019241426A1 (en) 2018-06-13 2019-12-19 Novartis Ag Bcma chimeric antigen receptors and uses thereof
US20210139549A1 (en) 2018-06-14 2021-05-13 Ajinomoto Co., Inc. Compound having affinity substance to antibody, cleavable portion, and reactive group, or salt thereof
WO2019240287A1 (en) 2018-06-14 2019-12-19 味の素株式会社 Compound comprising substance having affinity for antibody, cleavage site and reactive group, or salt thereof
US11548947B2 (en) 2018-06-21 2023-01-10 Regeneron Pharmaceuticals, Inc. Bispecific anti-PSMA X anti-CD28 antibodies and uses thereof
WO2020011706A1 (en) 2018-07-09 2020-01-16 Oslo Universitetssykehus Hf Two chimeric antigen receptors specifically binding cd19 and igkappa
WO2020014366A1 (en) 2018-07-10 2020-01-16 Intrexon Corporation Ror-1 specific chimeric antigen receptors and uses thereof
US20200040101A1 (en) 2018-07-18 2020-02-06 Askgene Pharma Inc. Novel antibodies and methods for making and using the same
US11384153B2 (en) 2018-07-19 2022-07-12 Regeneran Pharmaceuticals, Inc. Bispecific anti-BCMA x anti-CD3 antibodies and uses thereof
WO2020020194A1 (en) 2018-07-24 2020-01-30 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Gd2-based chimeric antigen receptor and application thereof
US11034771B2 (en) 2018-07-25 2021-06-15 I-Mab Biopharma Us Limited Anti-CD73 anti-PD-L1 bispecific antibodies
WO2020025792A1 (en) 2018-08-03 2020-02-06 Amgen Research (Munich) Gmbh Antibody constructs for cldn18.2 and cd3
US20220218746A1 (en) 2018-08-24 2022-07-14 Shenzhen Pregene Biopharma Co. Ltd. Bcma chimeric antigen receptor based on single domain antibody and use thereof
WO2020038146A1 (en) 2018-08-24 2020-02-27 深圳普瑞金生物药业有限公司 Bcma chimeric antigen receptor based on single domain antibody and use thereof
WO2020055975A1 (en) 2018-09-11 2020-03-19 Washington University Anti-trem-2 agonist antibodies
WO2020060924A1 (en) 2018-09-17 2020-03-26 Dualogics, Llc Use of a cd4/cd8 bispecific antibody for the treatment of autoimmune/inflammatory disorders
US20210363245A1 (en) 2018-09-17 2021-11-25 The United States Of America,As Represented By The Secretary,Department Of Health And Human Services Bicistronic chimeric antigen receptors targeting cd19 and cd20 and their uses
US10844128B2 (en) 2018-09-20 2020-11-24 Lentigen Technology, Inc. Compositions and methods for treating cancer with anti-CD123 immunotherapy
WO2020065330A2 (en) 2018-09-27 2020-04-02 Autolus Limited Chimeric antigen receptor
US11530268B2 (en) 2018-10-09 2022-12-20 Sanofi Trispecific anti-CD38, anti-CD28, and anti-CD3 binding proteins and methods of use for treating viral infection
WO2020076105A1 (en) 2018-10-10 2020-04-16 주식회사 노벨티노빌리티 Novel anti-c-kit antibody
US11492402B2 (en) 2018-10-15 2022-11-08 Novartis Ag TREM2 stabilizing antibodies
WO2020092654A1 (en) 2018-10-30 2020-05-07 Magenta Therapeutics, Inc. Anti-cd45 antibodies and conjugates thereof
WO2020092854A2 (en) 2018-11-01 2020-05-07 Juno Therapeutics, Inc. Chimeric antigen receptors specific for g protein-coupled receptor class c group 5 member d (gprc5d)
WO2020098599A1 (en) 2018-11-12 2020-05-22 江苏恒瑞医药股份有限公司 Anti-cd73 antibody, antigen-binding fragment thereof and application thereof
WO2020102591A1 (en) 2018-11-14 2020-05-22 Rubryc Therapeutics, Inc. Cd25 antibodies
US11208482B2 (en) 2018-11-26 2021-12-28 Forty Seven, Inc. Humanized antibodies against c-Kit
US20200261503A1 (en) 2018-12-01 2020-08-20 Allogene Therapeutics, Inc. Chimeric antigen receptors targeting b-cell maturation antigen and methods of use thereof
WO2020121195A1 (en) 2018-12-10 2020-06-18 Mor Research Applications Trem2 antibodies and uses thereof
US11453721B2 (en) 2018-12-19 2022-09-27 Regeneran Pharmaceuticals, Inc. Bispecific anti-MUC16 x anti-CD28 antibodies and uses thereof
WO2020127618A1 (en) 2018-12-21 2020-06-25 F. Hoffmann-La Roche Ag Tumor-targeted agonistic cd28 antigen binding molecules
WO2020132190A1 (en) 2018-12-21 2020-06-25 Multitude Inc. Antibodies specific to muc18
US11608376B2 (en) 2018-12-21 2023-03-21 Hoffmann-La Roche Inc. Tumor-targeted agonistic CD28 antigen binding molecules
WO2020135201A1 (en) 2018-12-28 2020-07-02 四川科伦博泰生物医药股份有限公司 Antibody and use thereof
WO2020139956A1 (en) 2018-12-28 2020-07-02 Sparx Therapeutics Inc. Binding molecules specific for claudin 18.2, compositons and methods thereof, for treatment of cancer and other diseases
WO2020143836A1 (en) 2019-01-11 2020-07-16 上海开拓者生物医药有限公司 Cd73 antibody, preparation method therefor and application thereof
WO2020143710A1 (en) 2019-01-11 2020-07-16 康诺亚生物医药科技（成都）有限公司 Anti-cd73 monoclonal antibody and application thereof
WO2020147321A1 (en) 2019-01-17 2020-07-23 Beijing Mabworks Biotech Co. Ltd. Antibodies binding human claudin 18.2 and uses thereof
WO2020148677A1 (en) 2019-01-18 2020-07-23 Janssen Biotech, Inc. Gprc5d chimeric antigen receptors and cells expressing the same
WO2020154293A1 (en) 2019-01-22 2020-07-30 Bristol-Myers Squibb Company Antibodies against il-7r alpha subunit and uses thereof
WO2020159754A2 (en) 2019-01-28 2020-08-06 Multitude Inc. Antibodies specific to cd44
WO2020160242A1 (en) 2019-02-01 2020-08-06 Regeneron Pharmaceuticals, Inc. Anti-il2 receptor gamma antigen-binding proteins
US20230248842A1 (en) 2019-03-08 2023-08-10 AbTis Co., Ltd. Site-specific antibody conjugation and antibody-drug conjugate as specific embodiment thereof
WO2020184944A1 (en) 2019-03-08 2020-09-17 앱티스 주식회사 Site-specific antibody conjugation and antibody-drug conjugate as specific example thereof
US10934337B2 (en) 2019-03-15 2021-03-02 Cartesian Therapeutics, Inc. Anti-BCMA chimeric antigen receptors
WO2020190737A1 (en) 2019-03-15 2020-09-24 Cartesian Therapeutics, Inc. Anti-bcma chimeric antigen receptors
US20200297634A1 (en) 2019-03-19 2020-09-24 Arcturus Therapeutics, Inc. Method of making lipid-encapsulated rna nanoparticles
WO2020198531A2 (en) 2019-03-26 2020-10-01 The Regents Of The University Of California Chimeric antigen receptor modified t-cells (car-t) for the treatment of hematological and solid tumor cancers
WO2020212710A1 (en) 2019-04-18 2020-10-22 Kymab Limited Antagonists anti-cd7 antibodies
WO2020216697A1 (en) 2019-04-23 2020-10-29 Innate Pharma Cd73 blocking antibodies
WO2020219770A1 (en) 2019-04-24 2020-10-29 Magenta Therapeutics, Inc. Anti-cd117 antibodies and uses thereof
US11203643B2 (en) 2019-05-10 2021-12-21 Lyvgen Biopharma Holdings Limited Humanized anti-CD137 antibodies and uses thereof
US11174319B2 (en) 2019-06-06 2021-11-16 Jacobio Pharmaceuticals Co., Ltd. Binding molecule specific for CD73 and use of binding molecule
WO2020253568A1 (en) 2019-06-19 2020-12-24 海正生物制药有限公司 Anti-cd73 antibody and application thereof
WO2020254827A1 (en) 2019-06-21 2020-12-24 Vhsquared Limited Polypeptides
WO2021008463A1 (en) 2019-07-12 2021-01-21 明济生物制药（北京）有限公司 Cldn18.2 antibody and use thereof
WO2021017892A1 (en) 2019-07-26 2021-02-04 上海复宏汉霖生物技术股份有限公司 Method and composition for anti-cd73 antibodies and variants
WO2021022304A2 (en) 2019-07-30 2021-02-04 Qlsf Biotherapeutics, Inc. MULTISPECIFIC BINDING COMPOUNDS THAT BIND TO LRRC15 AND CD3ε
WO2021018925A1 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021018859A2 (en) 2019-07-31 2021-02-04 F. Hoffmann-La Roche Ag Antibodies binding to gprc5d
WO2021027850A1 (en) 2019-08-12 2021-02-18 I-Mab Biopharma Co., Ltd. Anti-claudin 18.2 and anti-4-1bb bispecific antibodies and uses thereof
WO2021032157A1 (en) 2019-08-20 2021-02-25 Mabspace Biosciences (Suzhou) Co., Limited Novel anti-cldn18.2 antibodies
WO2021032173A1 (en) 2019-08-21 2021-02-25 Harbour Biomed (Shanghai) Co., Ltd. Anti-cd73 antibody and application thereof
WO2021044008A1 (en) 2019-09-04 2021-03-11 Universität Zürich Bispecific binding agent that binds to CD117/c-KIT and CD3
WO2021046159A1 (en) 2019-09-04 2021-03-11 Genentech, Inc. Cd8 binding agents and uses thereof
US11634500B2 (en) 2019-09-06 2023-04-25 Les Laboratoires Servier Anti-CD73 antibodies and compositions
WO2021044039A1 (en) 2019-09-06 2021-03-11 Scancell Limited Ssea-4 binding members
WO2021055437A1 (en) 2019-09-16 2021-03-25 The General Hospital Corporation Cd70 targeted chimeric antigen receptor (car) t cells and uses thereof
US20210087295A1 (en) 2019-09-23 2021-03-25 The Trustees Of The University Of Pennsylvania Disrupting tumor tissues by targeting fibroblast activation protein (fap)
WO2021072312A1 (en) 2019-10-11 2021-04-15 The Trustees Of The University Of Pennsylvania Compositions and methods for targeting cd13 and tim-3 with car t cells to treat acute myeloid leukemia
WO2021076564A1 (en) 2019-10-15 2021-04-22 Dragonfly Therapeutics, Inc. Proteins binding nkg2d, cd16 and flt3
WO2021091945A1 (en) 2019-11-05 2021-05-14 Janssen Biotech, Inc. Bcma-targeted car-t cell therapy of multiple myeloma
WO2021097223A2 (en) 2019-11-15 2021-05-20 Genzyme Corporation Biparatopic cd73 antibodies
WO2021099418A1 (en) 2019-11-19 2021-05-27 International - Drug - Development - Biotech Anti-cd117 antibodies and methods of use thereof
WO2021101823A1 (en) 2019-11-22 2021-05-27 Eli Lilly And Company Trem2 antibodies and uses thereof
WO2021102332A1 (en) 2019-11-22 2021-05-27 Silverback Therapeutics, Inc. Tgfbetar2 inhibitor-lrrc15 antibody conjugates and uses thereof
WO2021107566A1 (en) 2019-11-25 2021-06-03 주식회사 노벨티노빌리티 Antibody against c-kit and use thereof
US20230002489A1 (en) 2019-11-26 2023-01-05 Shanghai Epimab Biotherapeutics Co., Ltd. Antibodies to cd3 and bcma, and bispecific binding proteins made therefrom
US11919965B2 (en) 2019-12-06 2024-03-05 Regeneron Pharmaceuticals, Inc. Methods of treating multiple myeloma with bispecific anti-BCMA x anti-CD3 antibodies
WO2021111003A1 (en) 2019-12-06 2021-06-10 SOTIO a.s. Humanized cldn18.2 antibodies
WO2021118955A1 (en) 2019-12-13 2021-06-17 Aggamin Llc Methods and systems for treating or preventing pregnancy-related hypertensive disorders
WO2021127088A1 (en) 2019-12-18 2021-06-24 Janssen Biotech, Inc. Materials and methods for in vivo biological targeting
WO2021129765A1 (en) 2019-12-27 2021-07-01 Nanjing Legend Biotech Co., Ltd. Claudin18.2 binding moieties and uses thereof
WO2021138378A1 (en) 2019-12-30 2021-07-08 Obi Pharma, Inc. Antibodies, pharmaceutical compositions and uses thereof
WO2021138264A1 (en) 2019-12-30 2021-07-08 Seagen Inc. Methods of treating cancer with nonfucosylated anti-cd70 antibodies
WO2021138467A1 (en) 2020-01-03 2021-07-08 Incyte Corporation Anti-cd73 antibodies and uses thereof
US11124567B2 (en) 2020-01-13 2021-09-21 Denali Therapeutics Inc. Anti-TREM2 antibodies and methods of use thereof
WO2021155071A1 (en) 2020-01-29 2021-08-05 Inhibrx, Inc. Cd28 single domain antibodies and multivalent and multispecific constructs thereof
WO2021160267A1 (en) 2020-02-13 2021-08-19 UCB Biopharma SRL Bispecific antibodies against cd9 and cd7
WO2021160269A1 (en) 2020-02-13 2021-08-19 UCB Biopharma SRL Anti cd44-ctla4 bispecific antibodies
US20230075244A1 (en) 2020-02-20 2023-03-09 The Feinstein Institutes For Medical Research Agonist antibodies against endoglin and uses thereof
US20230144142A1 (en) 2020-02-27 2023-05-11 McSAF Inside Oncology Anti-cd56 antibody-drug conjugates and their use in therapy
WO2021174198A1 (en) 2020-02-28 2021-09-02 The Brigham And Women's Hospital, Inc. Selective modulation of transforming growth factor beta superfamily signaling via multi-specific antibodies
US20230067770A1 (en) 2020-02-28 2023-03-02 Shanghai Henlius Biotech, Inc. Anti-cd137 constructs, multispecific antibody and uses thereof
WO2021178896A1 (en) 2020-03-06 2021-09-10 Go Therapeutics, Inc. Anti-glyco-cd44 antibodies and their uses
WO2021188590A2 (en) 2020-03-16 2021-09-23 Magenta Therapeutics, Inc. T-cell bispecific binding proteins
WO2021186056A1 (en) 2020-03-20 2021-09-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Chimeric antigen receptor specific for human cd45rc and uses thereof
WO2021202642A2 (en) 2020-04-01 2021-10-07 The Regents Of The University Of California Immunotheranostic agent targeting mesenchymal stem cell-derived cancer cells and mesenchymal stem cell associated disease
WO2021205383A1 (en) 2020-04-09 2021-10-14 Aprilbio Co., Ltd. Monoclonal antibodies and antigen binding fragments thereof for suppressing cd73 immune checkpoint and uses thereof
WO2021213466A1 (en) 2020-04-22 2021-10-28 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
WO2021222227A1 (en) 2020-04-27 2021-11-04 Memorial Sloan-Kettering Cancer Center Chimeric antigen receptors targeting cd127 and use thereof
WO2021218874A1 (en) 2020-04-27 2021-11-04 启愈生物技术(上海)有限公司 Bispecific antibody targeting human claudin and human pdl1 proteins, and application thereof
WO2021227307A1 (en) 2020-05-12 2021-11-18 普米斯生物技术(珠海)有限公司 Anti-cd73 antibody and use thereof
WO2021241729A1 (en) 2020-05-29 2021-12-02 ブライトパス・バイオ株式会社 Anti-cd73 antibody and use thereof
WO2021245603A1 (en) 2020-06-04 2021-12-09 Crispr Therapeutics Ag Anti-cd70 antibodies and uses thereof
WO2021254481A1 (en) 2020-06-19 2021-12-23 信达生物制药(苏州)有限公司 Anti-claudin18.2 antibody and use thereof
WO2021259199A1 (en) 2020-06-22 2021-12-30 信达生物制药(苏州)有限公司 Anti-cd73 antibody and use thereof
WO2022002019A1 (en) 2020-06-30 2022-01-06 江苏恒瑞医药股份有限公司 Anti-cd70 antibody and application thereof
WO2022007808A1 (en) 2020-07-06 2022-01-13 康诺亚生物医药科技（成都）有限公司 Antibody binding to claudin-18.2 and use thereof
WO2022022718A1 (en) 2020-07-31 2022-02-03 北京市神经外科研究所 Anti-cd133 single-chain antibody and use thereof in preparation of drug for treating tumor
WO2022022720A1 (en) 2020-07-31 2022-02-03 北京市神经外科研究所 Anti-cd44 single-chain antibody and use thereof in preparing drug for treating tumor
US20230272089A1 (en) 2020-08-05 2023-08-31 Synthekine, Inc Il2rg binding molecules and methods of use
WO2022032293A2 (en) 2020-08-05 2022-02-10 Vigil Neuroscience, Inc. Treatment of diseases related to colony-stimulating factor 1 receptor dysfunction using trem2 agonists
US20230272090A1 (en) 2020-08-05 2023-08-31 Sandro Vivona Il2rb binding molecules and methods of use
WO2022037531A1 (en) 2020-08-17 2022-02-24 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
US11591401B2 (en) 2020-08-19 2023-02-28 Xencor, Inc. Anti-CD28 compositions
US20230193292A1 (en) 2020-08-20 2023-06-22 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Bcma-binding single variable structural domain and antigen-binding molecule
WO2022040577A1 (en) 2020-08-20 2022-02-24 City Of Hope Compositions and uses of cd45 targeted chimeric antigen receptor modified t cells
WO2022040608A1 (en) 2020-08-21 2022-02-24 City Of Hope Anti-cd5 antibody compositions and uses thereof
WO2022040088A2 (en) 2020-08-21 2022-02-24 Synthekine, Inc. Cd45 binding molecules and methods of use
US20230331872A1 (en) 2020-08-25 2023-10-19 Cytoimmune Therapeutics, Inc. Bispecific antibody car cell immunotherapy
WO2022056199A1 (en) 2020-09-11 2022-03-17 Janssen Biotech, Inc. Multi-specific immune targeting molecules and uses thereof
WO2022063853A1 (en) 2020-09-25 2022-03-31 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates and uses thereof
WO2022064191A1 (en) 2020-09-25 2022-03-31 Ucl Business Ltd Anti-cd45 antibodies and related therapeutics
WO2022067394A1 (en) 2020-09-30 2022-04-07 Newsouth Innovations Pty Limited Antigen-binding fragments and uses thereof
WO2022068854A1 (en) 2020-09-30 2022-04-07 Nanjing GenScript Biotech Co., Ltd. Antibodies targeting human claudin 18.2 and uses thereof
WO2022078344A1 (en) 2020-10-12 2022-04-21 南京驯鹿医疗技术有限公司 Antibody and chimeric antigen receptor (car) binding to cd70, and application thereof
WO2022081516A1 (en) 2020-10-13 2022-04-21 Janssen Biotech, Inc. Bioengineered t cell mediated immunity, materials and other methods for modulating cluster of differentiation iv &/or viii
WO2022082073A2 (en) 2020-10-16 2022-04-21 Board Of Regents, The University Of Texas System Compositions and methods for muc18 targeting
WO2022083049A1 (en) 2020-10-19 2022-04-28 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
WO2022083723A1 (en) 2020-10-23 2022-04-28 中山康方生物医药有限公司 Anti-cd73 antibody and use thereof
WO2022095803A1 (en) 2020-11-03 2022-05-12 南京北恒生物科技有限公司 Cd7-targeting humanized antibody and use thereof
WO2022096020A1 (en) 2020-11-09 2022-05-12 江苏中新医药有限公司 Anti-ecto-5'-nucleotidase antibody sequence
WO2022104009A1 (en) 2020-11-13 2022-05-19 Rubryc Therapeutics, Inc. Cd25 antibodies
WO2022105881A1 (en) 2020-11-20 2022-05-27 Bliss Biopharmaceutical (Hangzhou) Co., Ltd. Anti-cd73 antibody, antibody-drug conjugate, and use thereof
WO2022105914A1 (en) 2020-11-23 2022-05-27 江苏先声药业有限公司 Antibody binding to cd70 and application thereof
WO2022111616A1 (en) 2020-11-30 2022-06-02 石药集团巨石生物制药有限公司 Anti-cldn18.2 antibody, drug conjugate, and preparation method therefor and use thereof
WO2022125837A1 (en) 2020-12-09 2022-06-16 City Of Hope Compositions and uses of cd19 targeted chimeric antigen receptor modified immune cells
WO2022022749A1 (en) 2020-12-16 2022-02-03 南通大学 Use of cd44 antibody in preparation of medicine for treating parkinson's disease
WO2022127844A1 (en) 2020-12-17 2022-06-23 江苏先声药业有限公司 Cd5 antibody and use thereof
WO2022136888A1 (en) 2020-12-23 2022-06-30 Bivictrix Limited Bispecific antibodies binding to cd7 and cd33
WO2022147338A1 (en) 2020-12-30 2022-07-07 Ascendo Biotechnology, Inc. Monoclonal antibody against human mac-1 and uses thereof
WO2022143951A1 (en) 2020-12-31 2022-07-07 康诺亚生物医药科技（成都）有限公司 Development and use of function-enhanced antibody blocking agent
WO2022151851A1 (en) 2021-01-12 2022-07-21 上海雅科生物科技有限公司 Cd7-targeted engineered immune cell, chimeric antigen receptor, cd7 blocking molecule and use thereof
CN114763383A (en) 2021-01-13 2022-07-19 博生吉医药科技(苏州)有限公司 Monoclonal antibody targeting human BCMA and application thereof
WO2022155608A2 (en) 2021-01-15 2022-07-21 The General Hospital Corporation Cd64 chimeric receptor and uses thereof
WO2022159737A1 (en) 2021-01-22 2022-07-28 Celldex Therapeutics, Inc. Anti-kit antibodies and uses thereof
WO2022157094A2 (en) 2021-01-22 2022-07-28 Bayer Aktiengesellschaft Lrrc15 antibodies and conjugates thereof
US20220233591A1 (en) 2021-01-25 2022-07-28 Beijing Daxi Biotechnology Co., Ltd Preparation method of nk cells and use thereof in the treatment of cancer
WO2022161314A1 (en) 2021-01-27 2022-08-04 信达生物制药(苏州)有限公司 Single-domain antibody against cd16a and use thereof
WO2022166802A1 (en) 2021-02-04 2022-08-11 上海交通大学 Anti-human cd271 monoclonal antibody
WO2022175255A2 (en) 2021-02-16 2022-08-25 Janssen Pharmaceutica Nv Trispecific antibody targeting bcma, gprc5d, and cd3
WO2022174813A1 (en) 2021-02-19 2022-08-25 信达生物制药(苏州)有限公司 Anti-gprc5d×bcma×cd3 trispecific antibody and use thereof
WO2022179039A1 (en) 2021-02-24 2022-09-01 苏州近岸蛋白质科技股份有限公司 Anti-human cd73 antibody and use thereof
WO2022187050A1 (en) 2021-03-01 2022-09-09 BioLegend, Inc. Anti-cd117 agents and compositions and methods for making and using the same
WO2022212848A1 (en) 2021-04-02 2022-10-06 Teneobio, Inc. Agonistic anti-il-2r antibodies and methods of use
US11535869B2 (en) 2021-04-08 2022-12-27 Sana Biotechnology, Inc. CD8-specific antibody constructs and compositions thereof
WO2022214677A1 (en) 2021-04-09 2022-10-13 Cancer Research Technology Limited Anti-cd73 antibodies
WO2022221409A1 (en) 2021-04-14 2022-10-20 Villaris Therapeutics, Inc. Anti-cd122 antibodies and uses thereof
WO2022222910A1 (en) 2021-04-22 2022-10-27 南京北恒生物科技有限公司 Gprc5d-targeting antibody and use thereof
WO2022226317A1 (en) 2021-04-23 2022-10-27 Profoundbio Us Co. Anti-cd70 antibodies, conjugates thereof and methods of using the same
WO2022241082A1 (en) 2021-05-14 2022-11-17 Genentech, Inc. Agonists of trem2
WO2022243838A1 (en) 2021-05-18 2022-11-24 Janssen Biotech, Inc. Compositions comprising a t cell redirection therapeutic and an anti-cd44 therapeutic
WO2022242758A1 (en) 2021-05-21 2022-11-24 百奥泰生物制药股份有限公司 Anti-cd73 antibody and use thereof
WO2022247756A1 (en) 2021-05-23 2022-12-01 上海邦耀生物科技有限公司 Chimeric antigen receptor targeting gprc5d, and use thereof
WO2022247804A1 (en) 2021-05-23 2022-12-01 上海祥耀生物科技有限责任公司 Anti-gprc5d antibody, preparation method therefor, and use thereof
WO2022257835A1 (en) 2021-06-08 2022-12-15 Beijing Meikang Geno-Immune Biotechnology Co., Ltd. Cd7-based humanized chimeric antigen receptor and use thereof
WO2022262101A1 (en) 2021-06-17 2022-12-22 南京蓝盾生物科技有限公司 Anti-cd70 antibody having enhanced adcc effect and application thereof
WO2022263855A1 (en) 2021-06-18 2022-12-22 Autolus Limited Anti-cd307e single-domain antibodies, uses thereof in car t-cell and for the treatment of diseases
WO2023274183A1 (en) 2021-06-29 2023-01-05 江苏先声药业有限公司 Cd16 antibody and use thereof
WO2023278520A1 (en) 2021-06-30 2023-01-05 Board Of Regents, The University Of Texas System Polypeptides targeting cd70-positive cancers
WO2023280880A1 (en) 2021-07-05 2023-01-12 Trion Research Gmbh Multispecific antibodies binding to tumor-associated antigens causing no or reduced nerve pain in the treatment of cancer
WO2023004304A1 (en) 2021-07-20 2023-01-26 Inhibrx, Inc. Cd8-binding polypeptides and uses thereof
WO2023012802A1 (en) 2021-08-05 2023-02-09 Yeda Research And Development Co. Ltd. Antibodies for treating cancer
WO2023026791A1 (en) 2021-08-27 2023-03-02 株式会社免疫生物研究所 Antibody fragment specific to c-kit positive tumors
WO2023031403A1 (en) 2021-09-02 2023-03-09 F. Hoffmann-La Roche Ag Antibodies for the treatment of aml
WO2023039450A2 (en) 2021-09-07 2023-03-16 Duke University Compositions and methods for treatment of retinal degeneration
WO2023039612A1 (en) 2021-09-13 2023-03-16 The Board Of Regents Of The University Of Texas System Trem2 antigen binding proteins and uses thereof
WO2023037015A1 (en) 2021-09-13 2023-03-16 Universiteit Gent Aminopeptidase n-specific monoclonal antibodies and uses thereof
WO2023047100A1 (en) 2021-09-21 2023-03-30 Quell Therapeutics Ltd Anti-trem2 chimeric antigen receptor
US11613584B1 (en) 2021-10-13 2023-03-28 Immuneonco Biopharmaceuticals (Shanghai) Inc. Antibodies binding CD70, preparation and use thereof
WO2023078113A1 (en) 2021-11-02 2023-05-11 Epimab Biotherapeutics (Hk) Limited Anti-cd122 antibodies, anti-cd132 antibodies, and related bispecific binding proteins
WO2023078382A1 (en) 2021-11-05 2023-05-11 正大天晴药业集团股份有限公司 Antibody binding to gprc5d and use thereof
WO2023125728A1 (en) 2021-12-31 2023-07-06 康源博创生物科技(北京)有限公司 Anti-gprc5d antibody and use thereof
WO2023143537A1 (en) 2022-01-29 2023-08-03 恺兴生命科技(上海)有限公司 Gprc5d antibody and use thereof
WO2023183927A2 (en) 2022-03-24 2023-09-28 Actinium Pharmaceuticals, Inc. Humanized anti-cd45 antibodies
WO2023196445A1 (en) 2022-04-05 2023-10-12 Capstan Therapeutics, Inc. Peg-lipids and lipid nanoparticles
WO2023201316A2 (en) 2022-04-13 2023-10-19 Board Of Regents, The University Of Texas System Anti-soluble interleukin-7 receptor antibody therapy to treat autoimmune diseases
US20240034816A1 (en) 2022-05-27 2024-02-01 Sanofi NATURAL KILLER (NK) CELL ENGAGERS BINDING TO NKp46 AND BCMA VARIANTS WITH FC-ENGINEERING
WO2023235772A2 (en) 2022-05-31 2023-12-07 Fred Hutchinson Cancer Center Humanized anti-cd45 antibodies and uses thereof
WO2023236889A1 (en) 2022-06-06 2023-12-14 山东先声生物制药有限公司 Multi-specific antibody targeting bcma, gprc5d and t cells and application thereof
WO2024031091A2 (en) 2022-08-05 2024-02-08 Juno Therapeutics, Inc. Chimeric antigen receptors specific for gprc5d and bcma
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024046239A1 (en) 2022-08-30 2024-03-07 苏州缔码生物科技有限公司 Recombinant humanized monoclonal antibody targeting human gprc5d and application thereof
WO2024047558A2 (en) 2022-08-31 2024-03-07 Immuneel Therapeutics Private Limited Antigen-binding proteins and chimeric antigen receptors specific for domain 9 of human cd307e
WO2024064771A1 (en) 2022-09-20 2024-03-28 Vor Biopharma Inc. Anti-cd45-ign antibody drug conjugates and uses thereof
WO2024073723A2 (en) 2022-09-30 2024-04-04 Forte Subsidiary, Inc. Anti-cd122 antibodies and uses thereof
WO2024079015A1 (en) 2022-10-10 2024-04-18 F. Hoffmann-La Roche Ag Combination therapy of a gprc5d tcb and imids
US20240228610A9 (en) 2022-10-10 2024-07-11 Medimmune, Llc Compositions and methods of treating cancer with chimeric antigen receptors targeting claudin 18.2
WO2024081729A2 (en) 2022-10-12 2024-04-18 Mythic Therapeutics, Inc. Lrrc-15-binding protein constructs and uses thereof
WO2024086190A1 (en) 2022-10-18 2024-04-25 Kite Pharma, Inc. Novel cd19 binders, car-t constructs comprising the same, and methods of using the same
WO2024102770A1 (en) 2022-11-07 2024-05-16 Tessera Therapeutics, Inc. Lipid nanoparticle drug conjugates
WO2024131962A1 (en) 2022-12-23 2024-06-27 成都恩沐生物科技有限公司 Novel anti-gprc5d antibody
WO2024158047A1 (en) 2023-01-27 2024-08-02 第一三共株式会社 Anti-lrrc15 antibody
WO2024249954A1 (en) 2023-05-31 2024-12-05 Capstan Therapeutics, Inc. Lipid nanoparticle formulations and compositions

Non-Patent Citations (53)

* Cited by examiner, † Cited by third party
Title
"Introduction to Modern Liquid Chromatography", 1979, JOHN WILEY AND SONS
"The Peptides", vol. 3, 1981, ACADEMIC PRESS
"Thin Layer Chromatography", 1969, SPRINGER-VERLAG
ANZALONE ET AL., NATURE BIOTECHNOLOGY, vol. 38, 2020, pages 824 - 844
BINDER ET AL., FRONT IMMUNOL., vol. 9, 2020, pages 1090
BIOORG. MED. CHEM. LETT., vol. 24, 2014, pages 2855
CARPENTER ET AL., CLIN. CANCER RES., vol. 19, no. 8, 2013, pages 2048 - 2060
CHEM. EUR. J., vol. 16, 2010, pages 4037
ELKINS ET AL., MOL. CANCER THER., vol. 11, no. 10, 2012, pages 2222 - 2232
ENDSLEYHO, J., ACQUIR. IMMUNE DEFIC. SYNDR., vol. 61, 2012, pages 417
EVANS, AUSTRALIAN J. CHEM., vol. 60, no. 6, 2007, pages 384
FRANCO ET AL., J. IMMUNOL., vol. 190, no. 11, 2013, pages 5739 - 5746
FRIEDMAN ET AL., HUM. GENE THER., vol. 29, no. 5, 2018, pages 585 - 601
FUJII ET AL., BIOCONJUGATE CHEM., vol. 34, 2023, pages 728
FURNISSHANNAFORDSMITHTATCHELLL: "Vogel's A Textbook of Practical Organic Chemistry", INCLUDING QUALITATIVE ORGANIC ANALYSIS, 1989
GILLERON ET AL., NAT. BIOTECHNOL., vol. 31, 2013, pages 638 - 646
H.-D. JAKUBKEH. JESCHEIT: "Aminosauren, Peptide, Proteine", 1982, VERLAG CHEMIE
ISE ET AL., CLIN. CANCER RES., vol. 11, no. 1, 2005, pages 87 - 96
ISE ET AL., CLIN. CHEM. LAB. MED., vol. 44, no. 5, 2006, pages 594 - 602
J. AM. CHEM. SOC., vol. 132, 2010, pages 15790
J. AM. CHEM. SOC., vol. 133, 2011, pages 20288
J. F. W. MCOMIE: "Protective Groups in Organic Chemistry", 1973, PLENUM PRESS
J. ORG. CHEM., vol. 57, 1992, pages 4746
JOCHEN LEHMANN: "Chemie der Kohlenhydrate: Monosaccharide and Derivate", vol. 15, 1974, GEORG THIEME VERLAG
KOLB ET AL., ANGEWANDTE CHEMIE, vol. 40, no. 11, 2001, pages 2004
LAM ET AL., NAT. COMMUN., vol. 11, no. 1, 2020, pages 283
LI ET AL., J MOL BIOL., vol. 263, 1996, pages 209 - 26
MAILANKODY ET AL., N ENGL J MED., vol. 387, no. 13, 2022, pages 1196 - 1206
MATSUDA ET AL., MOL. PHARMACEUTICS, vol. 18, 2021, pages 4058
MOLINER-MORRO ET AL., BIOMOLECULES, vol. 10, no. 12, 2020, pages 1661
MUNSON ET AL., COMMUN. BIOL., vol. 4, 2021, pages 211 - 224
ORG. CHEM. FRONTIERS, vol. 4, 2017, pages 1819
ORG. PROC. RES. DEV., vol. 13, 2009, pages 478
ORG. PROC. RES. DEV., vol. 15, 2011, pages 515
PARHIZ ET AL., J. CONTROLLED RELEASE, vol. 291, 2018, pages 106 - 115
PILLARISETTI ET AL., BLOOD, vol. 135, 2020, pages 1232 - 43
POLSON ET AL., INT. IMMUNOL., vol. 18, no. 9, 2006, pages 1363 - 1373
RAMISHETTI ET AL., ACS NANO, vol. 9, 2015, pages 6706
RODRIGUEZ-OTERO ET AL., BLOOD CANCER J., vol. 14, no. 1, 2024, pages 24
SAKUMA, GENE AND GENOME EDITING, no. 100017, 2022, pages 3 - 4
SCATCHARD ET AL., ANN. N.Y. ACAD. SCI., vol. 51, 1949, pages 660
SMITH: "March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 2019, WILEY-INTERSCIENCE
STILLKAHNMITRA, J. ORG. CHEM., vol. 43, 1978, pages 2923 - 292
SYNTHESIS, vol. 11, 2005, pages 1829
TETRAHEDRON LETTERS, vol. 43, 2002, pages 7753
TETRAHEDRON, vol. 43, 1987, pages 45
TOMBACZ ET AL., MOLECULAR THERAPY, vol. 29, no. 11, 2021, pages 3293 - 3304
VEIGA ET AL., NAT. COMMS., vol. 9, 2018, pages 4493
WILKINSONHALE, MABS, vol. 14, no. 1, 2022, pages 2123299
WU ET AL., PROTEIN ENGINEERING, vol. 14, no. 12, 2001, pages 1025 - 1033
ZHANG ET AL., ISCIENCE, vol. 27, 2024, pages 109804
ZHAO ET AL., J. HEMATOL. ONCOL., vol. 11, no. 1, 2018, pages 141
ZHOU ET AL., MEDCOMM, vol. 3, no. 3, 2022, pages 155

Also Published As

Publication number Publication date
WO2025217454A3 (en) 2025-11-20

Similar Documents

Publication Publication Date Title
WO2024040194A1 (en) Conditioning for in vivo immune cell engineering
US20230320995A1 (en) Ionizable cationic lipids and lipid nanoparticles
US12311033B2 (en) Lipid nanoparticle formulations and compositions
US20220218614A1 (en) Ionizable cationic lipids and lipid nanoparticles, and methods of synthesis and use thereof
US20240002331A1 (en) Ionizable cationic lipids and lipid nanoparticles, and methods of synthesis and use thereof
US20250127728A1 (en) Constrained Ionizable Cationic Lipids and Lipid Nanoparticles
WO2025076113A1 (en) Ionizable cationic lipids with conserved spacing and lipid nanoparticles
EP3339320A1 (en) Chimeric antibody receptor to which anti-cotinine antibody is linked, and use thereof
JP2022538397A (en) Ultramodular IgG3-Based Spacer Domains and Multifunctional Sites for Implementation in Chimeric Antigen Receptor Design
WO2022151960A1 (en) B7-h3 chimeric antigen receptor-modified t cell and use thereof
US20210371491A1 (en) Chimeric antigen receptors that bind to prostate specific membrane antigen
US20250332281A2 (en) Humanized anti-cd8 antibodies and uses thereof
US20250161481A1 (en) Targeted lnp delivery
WO2025217454A2 (en) Ionizable cationic lipids and lipid nanoparticles
WO2025217452A1 (en) Constrained ionizable cationic lipids and lipid nanoparticles
US20250302763A1 (en) Immune engineering amplification
US20250144234A1 (en) RNA for In vivo Transfection with Increased Expression
US20230019107A1 (en) Reversibly inhibited binding molecules