WO2023159108A1

WO2023159108A1 - Cd2-binding and cd28-binding membrane-bound polypeptides and uses thereof for evading host immune response

Info

Publication number: WO2023159108A1
Application number: PCT/US2023/062717
Authority: WO
Inventors: Tom WICKHAM; Philippe KIEFFER-KWON; Ting Xi GUO; Gideon Gross; Hadas WEINSTEIN-MAROM
Original assignee: Gentibio, Inc.; Migal Galilee Research Instutite Ltd
Priority date: 2022-02-16
Filing date: 2023-02-16
Publication date: 2023-08-24

Abstract

Disclosed herein are compositions and methods for arming therapeutic cells with a membrane-bound polypeptide comprising an extracellular CD2-binding domain or CD28- binding domain and an extracellular elongation domain, which prevents immune synapse formation with host immune cells and deleterious toxic effects thereof. The disclosed compositions and methods thereby provide a mechanism by which host immune attack can be evaded.

Description

CD2-BINDING AND CD28-BINDING MEMBRANE-BOUND POLYPEPTIDES AND USES THEREOF FOR EVADING HOST IMMUNE RESPONSE

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/310,997, filed February 16, 2022, and U.S. provisional application number 63/310,991, filed February 16, 2022, each of which is incorporated by reference herein in its entirety.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (G097170026WO00-SEQ-JXVxml; Size: 53,571 bytes; and Date of Creation: February 13, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

Cell therapies have huge potential to treat disease, however they suffer from the challenge of being attacked by host immune cells after being administered to a subject.

SUMMARY

A common hurdle in cell therapy is that therapeutic cells are attacked by host immune cells (e.g., T cells), thereby compromising the intended therapeutic effect. The present disclosure provides compositions and methods for arming therapeutic cells with a mechanism by which host immune attack can be evaded. Such recognition and attack of a target cell (e.g., a therapeutic cell) by a T cell occurs through the formation, between the T cell and the therapeutic cell, of an immune synapse in which receptors on the surface of the T cell bind to receptors on the surface of the therapeutic cell. This disclosure is based, at least in part, on the development of membrane-bound polypeptides, for expression on a therapeutic cell, that act as decoy receptors in that they engage with T cell receptors involved in the immune synapse, in a manner that does not result in T cell attack upon the therapeutic cells. The membrane-bound polypeptides of the present disclosure engage with CD2 or CD28 on host immune cells (e.g., host T cells) via an extracellular CD2-binding domain or extracellular CD28 -binding domain and prevent immune synapse formation and deleterious toxic effects thereof via an elongation domain that prevents relevant members of immune synapse formation from engaging with each other. The present disclosure is also based on the realization that to be most effective as a mechanism for host immune evasion, membrane-bound polypeptides expressed on therapeutic cells that must provide a balanced avidity that is not so high as to cause aggregation of therapeutic cells with other cells expressing CD2 or CD28, or that is so low as to not being able to engage with host T cells at all. The present disclosure is further based on the development of certain features of membrane-bound polypeptides expressed on therapeutic cells that provide this balanced avidity. These features include a particular length of the extracellular portion of the polypeptide, a CD2- or CD28-binding domain that provides an affinity to CD2 or CD28 that is not so high as to bind to CD2 or CD28 outside the context of an immune synapse (e.g., to CD2 or CD28 expressed on endothelial cells) but high enough to engage with CD2 or CD28 in the context of an immune synapse, and a level of expression on a therapeutic cell that also controls the avidity. Thus, to be most effective in preventing the elimination of therapeutic cells by T cells, the membrane-bound polypeptides of the present disclosure are designed to promote binding to CD2 or CD28 expressed on T cells at the time of recognition of the therapeutic cell as a non-self cell, while reducing their binding to CD2 or CD28 outside the context of an immune reaction, thereby avoiding unnecessary cell aggregation and loss of therapeutic cells.

The disclosure, in some aspects, provides a membrane-bound polypeptide comprising:

(a) an extracellular CD2-binding domain comprising a modified CD2-binding domain of CD58, wherein the modified CD2-binding domain of CD58 comprises one or more amino acid substitutions relative to SEQ ID NO: 2, or a CD2-binding single-chain variable fragment (scFv);

(b) an extracellular elongation domain comprising at least one rigid protein module; and

(c) a transmembrane domain; wherein (a)-(c) are connected from N-terminus to C-terminus in the following order: transmembrane domain, extracellular elongation domain, and extracellular CD2-binding domain.

In some embodiments, the membrane -bound polypeptide further comprises (d) an intracellular domain. In some embodiments, (a)-(d) are connected from N-terminus to C- terminus in the following order: intracellular domain, transmembrane domain, extracellular elongation domain, and extracellular CD2-binding domain. In some embodiments, the extracellular CD2-binding domain is connected to the extracellular elongation domain by a linker. In some embodiments, one or more amino acid substitutions relative to SEQ ID NO: 2 are substitutions at E25, K29, K32, D33, E37 and/or R44 of SEQ ID NO: 2. In some embodiments, one or more amino acid substitutions relative to SEQ ID NO: 2 are E25A, K29A, K32A, D33A, E37A and/or R44A relative to SEQ ID NO: 2.

In some embodiments, the extracellular CD2-binding domain comprises any one of SEQ ID NOs: 3-8. In some embodiments, the membrane bound polypeptide further comprises a second extracellular CD2-binding domain comprising a modified CD2-binding domain of CD58, wherein the modified CD2-binding domain of CD58 comprises one or more amino acid substitutions relative to SEQ ID NO: 2.

In some embodiments, the at least one rigid protein module comprises an Ig-like domain of CD22.

In some embodiments, the extracellular elongation domain comprises a first rigid protein module and a second rigid protein module, wherein the first rigid protein module is N-term relative to the second rigid protein module, and wherein the first rigid protein module comprises a sequence that is at least 85% identical to the C6 Ig-like domain of CD22 as set forth in SEQ ID NO: 29, and the second rigid protein module comprises a sequence that is at least 85% identical to the C5 Ig-like domain of CD22 as set forth in SEQ ID NO: 28. In some embodiments, the extracellular elongation domain further comprises a third rigid protein module that is C-terminal (C-term) relative to the second rigid protein module, wherein the third rigid protein module comprises a sequence that is at least 85% identical to the C4 Ig-like domain of CD22 as set forth in SEQ ID NO: 27. In some embodiments, the extracellular elongation domain further comprises a fourth rigid protein module that is C-term relative to the third rigid protein module, wherein the fourth rigid protein module comprises a sequence that is at least 85% identical to the C3 Ig-like domain of CD22 as set forth in SEQ ID NO: 26. In some embodiments, the extracellular elongation domain further comprises a fifth rigid protein module that is C-term relative to the fourth rigid protein module, wherein the fifth rigid protein module comprises a sequence that is at least 85% identical to the C2 Ig-like domain of CD22 as set forth in SEQ ID NO: 25.

In some embodiments, the extracellular CD2-binding domain is linked to the extracellular elongation domain via a linker comprising the amino acid sequence of SEQ ID NO: 1.

The disclosure, in some aspects provides a membrane-bound polypeptide comprising: (a) an extracellular CD2-binding domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-8; (b) an extracellular elongation domain comprising at least one rigid protein module comprising an Ig-like domain of CD22; and

In some embodiments, the membrane -bound polypeptide, further comprises (d) an intracellular domain. In some embodiments, (a)-(d) are connected from N-terminus to C- terminus in the following order: intracellular domain, transmembrane domain, extracellular elongation domain, and extracellular CD2-binding domain.

In some embodiments, the extracellular CD2-binding domain comprises any one of SEQ ID NOs: 3-8. In some embodiments, the membrane-bound polypeptide further comprises a second extracellular CD2-binding domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-8.

The disclosure, in some aspects, provides a membrane-bound polypeptide comprising an extracellular CD2-binding domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-8; and a CD22 stalk comprising an extracellular elongation domain, a transmembrane domain, and optionally an intracellular domain sequence. In some embodiments, the CD22 stalk comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 9-12. In some embodiments, the extracellular CD2-binding domain is linked to the CD22 stalk directly, via a linker comprising the amino acid sequence of SEQ ID NO: 1, or via a CD8 linker. In some embodiments, the membrane-bound polypeptide comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 13-22.

The disclosure, in some embodiments, provides a nucleic acid comprising a sequence encoding any one of the membrane-bound polypeptides described herein.

The disclosure, in some embodiments, provides a vector comprising any one of the nucleic acids described herein. In some embodiments, the vector further comprises a promoter operably linked to the nucleic acid sequence encoding the membrane-bound polypeptide.

The disclosure, in some embodiments, provides a method for producing a therapeutic cell capable of evading a host immune system, the method comprising inserting into the cell the any one of the nucleic acids described herein, contacting the cell with any one of the vectors described herein, or expressing in the cell any one of the membrane -bound polypeptides described herein.

The disclosure, in some embodiments, provides a cell expressing any one of the membrane-bound polypeptides described herein, or comprising any one of the nucleic acids or vectors described herein. In some embodiments, the cell is a T cell, optionally a Treg. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) or a TCR.

The disclosure, in some embodiments, provides a method comprising administering to a subject any one of the cells described herein.

(a) an extracellular CD28-binding domain;

(c) a transmembrane domain, wherein (a)-(c) are connected from N-terminus to C-terminus in the following order: transmembrane domain, extracellular elongation domain, and extracellular CD28-binding domain. In some embodiments, the membrane bound polypeptide, further comprises (d) an intracellular domain. In some embodiments, (a)-(d) are connected from N-terminus to C- terminus in the following order: intracellular domain, transmembrane domain, extracellular elongation domain, and extracellular CD28-binding domain. In some embodiments, the extracellular CD28-binding domain is connected to the extracellular elongation domain by a linker.

In some embodiments, the extracellular CD28-binding domain comprises a CD28- binding domain of a B7 costimulatory ligand. In some embodiments, the B7 costimulatory ligand is CD80.

In some embodiments, the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD80 CD28-binding domain as set forth in SEQ ID NO: 34. In some embodiments, the B7 costimulatory ligand is CD86. In some embodiments, the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD 86 CD28 -binding domain as set forth in SEQ ID NO: 35.

In some embodiments, the at least one rigid protein module comprises an Ig-like domain of CD22. In some embodiments, the extracellular elongation domain comprises a first rigid protein module and a second rigid protein module, wherein the first rigid protein module is N-term relative to the second rigid protein module, and wherein the first rigid protein module comprises a sequence that is at least 85% identical to the C6 Ig-like domain of CD22 as set forth in SEQ ID NO: 29, and the second rigid protein module comprises a sequence that is at least 85% identical to the C5 Ig-like domain of CD22 as set forth in SEQ ID NO: 28.

In some embodiments, the extracellular elongation domain further comprises a third rigid protein module that is C-term relative to the second rigid protein module, wherein the third rigid protein module comprises a sequence that is at least 85% identical to the C4 Ig-like domain of CD22 as set forth in SEQ ID NO: 27.

In some embodiments, the extracellular elongation domain further comprises a fourth rigid protein module that is C-term relative to the third rigid protein module, wherein the fourth rigid protein module comprises a sequence that is at least 85% identical to the C3 Ig- like domain of CD22 as set forth in SEQ ID NO: 26. In some embodiments, the extracellular elongation domain further comprises a fifth rigid protein module that is C-term relative to the fourth rigid protein module, wherein the fifth rigid protein module comprises a sequence that is at least 85% identical to the C2 Ig-like domain of CD22 as set forth in SEQ ID NO: 25.

In some embodiments, the extracellular CD28-binding domain is linked to the extracellular elongation domain via a linker comprising the amino acid sequence of SEQ ID NO: 1.

In the disclosure, in some aspects, provides a membrane-bound polypeptide comprising an extracellular CD28-binding domain and a CD22 stalk comprising an extracellular elongation domain, a transmembrane domain, and optionally an intracellular domain sequence. In some embodiments, wherein the CD22 stalk comprises a sequence that is at least 85% identical to SEQ ID NO: 9.

In some embodiments, the extracellular CD28-binding domain comprises a CD28- binding domain of a B7 costimulatory ligand. In some embodiments, the B7 costimulatory ligand is CD80. In some embodiments, the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD80 CD28-binding domain as set forth in SEQ ID NO: 34. In some embodiments, the B7 costimulatory ligand is CD86. In some embodiments, the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD86 CD28-binding domain as set forth in SEQ ID NO: 35.

In some embodiments, the membrane bound polypeptides have a sequence identical to SEQ ID NO: 40 or 41.

The disclosure, in some embodiments, provides a nucleic acid comprising a sequence encoding the membrane-bound polypeptide described herein.

In some embodiments, the disclosure provides a vector comprising any one of the nucleic acids described herein. In some embodiments, the vector further comprises a promoter operably linked to the nucleic acid sequence encoding the membrane-bound polypeptide.

The disclosure, in some embodiments, provides a method for producing a therapeutic cell capable of evading a host immune system, the method comprising inserting into the cell any one of the nucleic acid of described herein, contacting the cell with any one of the vector described herein, or expressing in the cell the any one of the membrane-bound polypeptide described herein.

The disclosure, in some embodiments, provides a cell expressing any one of the membrane-bound polypeptide described herein, or comprising any one of the nucleic acids described herein or any one of the vectors described herein. In some embodiments, the cell is a T cell, optionally a Treg. In some embodiments, the T cell expresses a chimeric antigen receptor (CAR) or a TCR.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

FIG. 1 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig- like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 2 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 linked by a linker to an extracellular elongation domain comprising four rigid protein modules, C3-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 3 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 linked by a linker to an extracellular elongation domain comprising three rigid protein modules, C4-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 4 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 linked by a linker to an extracellular elongation domain comprising two rigid protein modules, C5-C6 Ig- like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 5 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 having the mutation E25A (indicated by an asterisk), linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 6 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 having the mutation K29A (indicated by an asterisk), linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 7 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 having the mutation K32A (indicated by an asterisk), linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 8 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 having the mutation D33A (indicated by an asterisk), linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 9 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 having the mutation E37A (indicated by an asterisk), linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 10 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 having the mutation R44A (indicated by an asterisk), linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 11 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD2-binding domain of CD58 having the mutation K32A (indicated by an asterisk), linked by a linker to an extracellular elongation domain comprising two rigid protein modules, C5-C6 Ig-like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 12 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD28-binding domain(s) of CD80 linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig- like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain.

FIG. 13 provides a schematic of a membrane-bound polypeptide as described in the present disclosure, comprising an extracellular CD28-binding domain(s) of CD86 linked by a linker to an extracellular elongation domain comprising five rigid protein modules, C2-C6 Ig- like domain of CD22. The arrow represents a transmembrane domain and/or an intracellular domain. FIG. 14 provides a schematic illustrating membrane-bound polypeptides as described in the present disclosure, comprising at least one extracellular CD2 binding domain (e.g., one extracellular CD2-binding domain of CD58, two extracellular CD2-binding domains of CD58, or an scFV domain) connected to an extracellular elongation domain either directly (no linker) or with a linker (e.g., a GGGS (G3S) (SEQ ID NO: 1) linker or a CD8 linker). Exemplary extracellular elongation domains shown in FIG. 14 include a C2-C6 Ig-like domain of CD22 (e.g., five rigid protein modules; left), CD45RO (middle), or CD45RABC (right). The arrow represents a transmembrane domain and/or an intracellular domain.

FIGs. 15A-15B show expression data of 293T cells transduced with the constructs depicted in FIG. 14 (see also, Table 2). FIG. 15A provides a graph of quantified expression levels of the constructs on 293T cells. FIG. 15B provides representative flow plots of CD22 expression of the l(+)22 CD2 IEE construct (see Table 2) and a control.

FIGs. 16A-16B demonstrate that construct expression on 293T cells reduces antigenspecific T cell responses. FIG. 16A provides a graph of normalized CD137 expression levels on CD8+ TCR-engineered T cells transfected with the constructs found in FIG. 14 (Table 2). FIG. 16B provides representative flow plots of CD8+ effector T cell responses.

FIGs. 17A-17B demonstrate construct expression on 293T cells reduced allogeneic T cell responses. FIG. 17A provides a graph of the quantified frequency of CD8+ CFSE-low cells transfected with the constructs found in FIG. 14 (Table 2). FIG. 17B provides representative plots with CFSE-low (lower band) and CFSE-high (upper band) gates.

DETAILED DESCRIPTION

An immune synapse is the interface between an antigen-presenting cell or target cell (e.g., a therapeutic cell administered to a subject) and an immune cell (e.g., T cell). The immune synapse is also known as the supramolecular activation cluster (SMAC), which comprises molecules involved in T cell activation. The SMAC is divided into the central SMAC (c-SMAC), peripheral SMAC (p-SMAC), and distal SMAC (d-SMAC). Examples of proteins in the c-SMAC include molecules such as the 9 isoform of protein kinase C, CD2, CD4, CD8, CD28, Lek, and Fyn. Examples of proteins in the p-SMAC include lymphocyte function-associated antigen- 1 (LFA-1) and the cytoskeletal protein talin. Examples of proteins in the d-SMAC include CD43 and CD45.

Immune synapse formation involves an initial interaction between LFA-1 present in the p-SMAC of a T cell and non-specific adhesion molecules (such as ICAM-1 or ICAM-2) on a target cell (e.g., therapeutic cell). When bound to a target cell (e.g., therapeutic cell), the T-cell can extend pseudopodia and scan the surface of the target cell (e.g., therapeutic cell) to find a specific peptide :MHC complex (e.g., specific peptide: HLA complex) to which the TCR binds. This binding initiates signal activation through formation of microclusters/lipid rafts. Specific signaling pathways lead to polarization of the T-cell by orienting its centrosome toward the site of the immune synapse and promoting clustering of TCRs and integrins.

Immune synapses are postulated to have several functions, including but not limited to the regulation of lymphocyte activation, the transfer of peptide-MHC complexes (e.g., peptide-HLA complexes) from APCs to lymphocytes, and directing secretion of cytokines or lytic granules.

In the context of cell therapy, the formation of an immune synapse between a therapeutic cell and a host immune cell (e.g., T cell) may lead to the recognition of the therapeutic cell as a non- self cell and to the elimination of the therapeutic cell via secretion by the T cell (e.g., cytotoxic CD8+ T cell) of cytolytic (e.g., apoptosis-inducing) enzymes. Elimination of the therapeutic cells attenuates the intended therapeutic effect, and jeopardizes the effectiveness of cell therapy.

Provided herein are compositions and methods for producing therapeutic cells, or any other target cell type, that evade host immune system attacks by expressing on their surface a membrane-bound polypeptide, i.e., a decoy receptor, that engages with a member of the SMAC on host immune cells and prevents (e.g., interferes with) the formation of immune synapses with the host immune cells, thereby evading host immune attack. The membranebound polypeptides of the present disclosure engage a member of the SMAC (e.g., cSMAC, p-SMAC, or d-SMAC) via an extracellular SMAC-binding domain (e.g., an extracellular CD2-binding domain and/or extracellular CD28-binding domain) and prevent immune synapse formation when expressed on a therapeutic cell via an extracellular elongation domain (also referred to herein as a stalk or a spacer) that is rigid by acting as a spacer so that when engaged with a member of the SMAC, other members of the SMAC are unable to engage with counterpart proteins on the therapeutic cell. Further, the membrane-bound polypeptides of the present disclosure provide a balanced avidity that is not so high as to cause aggregation of therapeutic cells with other cells expressing CD2 or CD28, or that is so low as to not being able to engage with host T cells at all, owning to certain structural features, e.g., length of the extracellular portion of the polypeptide, a CD2-binding domain that provides an affinity to CD2 that is not so high as to bind to CD2 outside the context of an immune synapse (e.g., to CD2 expressed on endothelial cells) but high enough to engage with CD2 in the context of an immune synapse or length of the extracellular portion of the polypeptide, a CD28-binding domain that provides an affinity to CD28 that is not so high as to bind to CD28 outside the context of an immune synapse (e.g., to CD28 expressed on endothelial cells) but high enough to engage with CD28 in the context of an immune synapse; and a level of expression on a therapeutic cell that also controls the avidity. Non-limiting examples of CD2 binding constructs are shown in FIGs. 1-11 and 14. Non-limiting examples of CD28 binding constructs are shown in FIGs. 12 and 13.

Membrane-bound polypeptides that prevent immune synapse formation

Provided herein is a membrane-bound polypeptide that can be expressed on the surface of a therapeutic cell such that it engages with a SMAC member on host immune cells, e.g., CD2 or CD28, and prevents immune synapse formation via an elongation domain that acts as a spacer.

In some embodiments, a membrane-bound polypeptide as provided herein comprises (a) an extracellular SMAC -binding domain, (b) an extracellular elongation domain comprising one or more rigid protein modules, and (c) a transmembrane domain that anchors the polypeptide to a cell (e.g., a therapeutic cell). In some embodiments, a membrane-bound polypeptide further comprises an intracellular domain. In some embodiments, a membranebound polypeptide further comprises a signal peptide. In some embodiments, the extracellular portion of the membrane -bound polypeptides of the present application comprises an extracellular SMAC -binding domain and an extracellular elongation domain comprising one or more rigid protein modules. In some embodiments, the extracellular portion of the membrane-bound polypeptides of the present application comprises an extracellular SMAC -binding domain, an extracellular elongation domain comprising one or more rigid protein modules, and one or more linkers (e.g., a linker linking the SMAC-binding domain and elongation domain, or a linker linking the SMAC-binding domain and transmembrane domain).

To be effective in preventing the elimination of therapeutic cells by T cells, the avidity of the membrane-bound polypeptides of the present disclosure when expressed on a target cell (e.g., a therapeutic cell) to bind to a SMAC member (e.g., CD2 or CD28) is such that the level of binding to the SMAC member expressed on a T cell at the time of recognition of the therapeutic cell as a non-self cell, thereby preventing the formation of the immune synapse between the T cell and the therapeutic cell, is greater (e.g., 1.5 times, 2 times, 3 times, 4, times, 5 times, 6 times, 7 times, 8 times, 9, times, or 10 or more times greater) than the level of binding to the SMAC member (e.g., CD2 or CD28) outside the context of an immune reaction to the presence of the therapeutic cell (e.g., on CD2- expressing dendritic cells on CD28-expressing dendritic cells, monocytes, NK cells, CD2 expressing T cells outside the context of being recognized by a TCR, or CD28 expressing T cells outside the context of being recognized by a TCR), thereby avoiding unnecessary cell aggregation. As used herein, avidity is defined as the tendency (e.g., likelihood) of cells to come together (e.g., aggregate) to form a stable interaction (e.g., an immune synapse). By shortening the length of the extracellular portion of the membrane-bound polypeptide (comprising the extracellular SMAC -binding domain and the extracellular elongation domain with any linkers between or adjacent to those domains), the avidity of the membrane-bound polypeptide as expressed on a target cell for binding a SMAC member is reduced due to inhibition by the glycocalyx on both the target cell and the host cells. Another means of tailoring avidity is to reduce the affinity of the extracellular SMAC-binding domain on the membrane-bound polypeptide, thereby making it harder for the membrane -bound polypeptide to bind randomly to the SMAC member; in this case, the initiation of an immune synapse would compensate for the lower binding affinity by providing proximity between the mutated extracellular SMAC-binding domain and the SMAC member. Yet another means of tailoring avidity is to adjust the expression level of the membrane-bound polypeptide on the surface of the therapeutic cells, for example by tailoring the number of copies of the membrane-bound polypeptide expressed on the surface of the therapeutic cell and/or through the use of appropriate promoters, optionally inducible/controllable promoters.

In some embodiments, a membrane-bound polypeptide as provided herein has the following arrangement of domains from N-term to C-term: transmembrane domain, extracellular elongation domain, and extracellular SMAC-binding domain. In some embodiments, a membrane-bound polypeptide as provided herein has the following arrangement of domains from N-term to C-term: intracellular domain, transmembrane domain, extracellular elongation domain, and extracellular SMAC-binding domain. In some embodiments, a membrane-bound polypeptide has the reverse arrangement wherein the C- terminal end of the polypeptide is intracellular or in the transmembrane domain rather than the extracellular binding domain. In some embodiments, any one of the domains in the membrane-bound polypeptide is connected to another domain via a linker.

Extracellular SMAC-binding domain The extracellular domain of a membrane-bound polypeptide as described herein comprises a domain that can engage with a member of SMAC on the surface of a host immune cell, i.e., a SMAC -binding domain. In some embodiments, a SMAC-binding domain is a CD2-binding domain. In some embodiments, a SMAC-binding domain is a CD28- binding domain.

In some embodiments, the extracellular CD2-binding domain comprises a human sequence. In some embodiments, extracellular CD2-binding domain comprises an antibody or antibody fragment thereof, e.g., an scFv. In some embodiments, the extracellular CD28- binding domain comprises a human sequence. In some embodiments, extracellular CD28- binding domain comprises an antibody or antibody fragment thereof, e.g., an scFv.

In some embodiments, the extracellular binding domain comprises a CD2-binding domain from LFA-3 (e.g., CD58 or CD48) or a functional fragment(s) thereof. In some embodiments, the CD2-binding domain from LFA-3 comprises a CD58 binding domain. In some embodiments, the CD2-binding domain from LFA-3 comprises two CD58 binding domains. In some embodiments the extracellular binding domain comprises an antibody or antibody fragment thereof, such as a scFV (e.g., an scFv derived from the VH and VL of a monoclonal antibody).

In some embodiments, the extracellular binding domain comprises a CD28-binding domain of a B7 costimulatory ligand (e.g., CD80 or CD86) or a functional fragment(s) thereof. In some embodiments, the B7 costimulatory ligand is CD80 (also referred to as B7- 1). In some embodiments, the B7 costimulatory ligand is CD86 (also referred to as B7-2). In some embodiments, the CD28-binding domain comprises an Ig-like V-type of CD80, or a functional fragment thereof. In some embodiments, the CD28-binding domains comprises an Ig-like C2-type of CD80, or a functional fragment thereof. In some embodiments, the CD28- binding domain comprises an Ig-like V-type of CD80, or a functional fragment thereof and an Ig-like C2-type of CD80, or a functional fragment thereof. In some embodiments, the CD28-binding domains comprises an Ig-like V-type of CD86, or a functional fragment thereof. In some embodiments, the CD28-binding domains comprises an Ig-like C2-type of CD86, or a functional fragment thereof. In some embodiments, the CD28-binding domains comprises an Ig-like V-type of CD86, or a functional fragment thereof and an Ig-like C2-type of CD86, or a functional fragment thereof.

As used herein, a functional fragment of a domain is one that comprises some fraction (e.g., 90%) of the consecutive amino acids that constitute the domain of which the fragment is a part, and which retains at least a measurable capacity (e.g., 50% capacity) to bind the relevant SMAC member (e.g., as measured by binding affinity) as that of the domain.

SEQ ID NO: 2 provides an example of a CD2-binding domain from LFA-3. In some embodiments, a functional fragment of a CD2-binding domain from LFA-3 comprises at least 10% (e.g., at least 10 %, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%) of the capacity to bind CD2 of that of CD2-binding domain of LFA-3 (e.g., as set forth in SEQ ID NO: 2). In some embodiments, a functional fragment of a CD2- binding domain from LFA-3 comprises at least 5 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 85, or at least 90) consecutive amino acids of the CD2-binding domain of LFA-3 (e.g., as set forth in SEQ ID NO: 2).

SEQ ID NOs: 34 and 35 provide examples of CD28-binding domains of a B7 costimulatory ligand. In some embodiments, a functional fragment of a CD28-binding domain of a B7 costimulatory ligand comprises at least 10% (e.g., at least 10 %, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%) of the capacity to bind CD28 of that of CD28-binding domain of a B7 costimulatory ligand (e.g., as set forth in SEQ ID NO: 34 or 35). In some embodiments, a functional fragment of a CD28-binding domain of a B7 costimulatory ligand comprises at least 5 (e.g., at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 85, or at least 90) consecutive amino acids of the CD28-binding domain of a B7 costimulatory ligand (e.g., as set forth in SEQ ID NO: 34 or 35).

In some embodiments, the extracellular CD2-binding domain of the presently described membrane -bound polypeptides has a capacity to bind CD2 that is lower (e.g., up to 1%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 7%, up to 8%, up to 9%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, or up to 50 % capacity; or by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90% or more lower; or at least 2 times, 3 times, 4, times, 5 times, 10 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 times, 140 times, or 150 times or more less) than the binding capacity to bind CD2 than that of LFA-3 or the CD2-binding domain of LFA-3 (e.g., having SEQ ID NO: 2). Such a reduced binding capacity, or affinity, to CD2, is helpful to reduce the avidity of the polypeptide as expressed on the target cell (e.g., a therapeutic cell) so that the therapeutic cell does not aggregate with CD2-expressing host cells outside the context of recognition for destruction by a host T cell. In some embodiments, an extracellular binding domain binds to CD2 with a dissociation constant (KD) of at least IO^-4 M (e.g., at least 10’⁴ M, at least 10’⁵ M, at least 10’⁶ M, at least 10’⁷ M; at least 10’⁸ M; at least 10’⁹ M; at least IO’¹⁰ M; at least 10’¹¹ M; at least 10’¹² M; or at least 10’¹³ M). In some embodiments, an extracellular binding domain binds to CD2 with a dissociation constant (KD) of at most 10’⁶ M (e.g., at least 10’⁶ M, at least 10’⁵ M, at least 10’⁴ M, at least 10’³ M; at least 10’² M; at least 10’¹ M; and at least 10° M. In some embodiments, an extracellular binding domain binds to CD2 with a dissociation constant (KD) of 10° M-10’⁶ M (e.g., 10° M-10’⁶ M, 10° M-10’⁵ M, 10° M-10"⁴ M, 10° M-10’³ M, 10’¹ M-10’⁶ M, 10’¹ M-10’⁵ M, 10’¹ M-10"⁴ M, 10’¹ M-10’³ M, 10’² M-10’⁶ M, 10’² M-10’⁵ M, 10’² M-10"⁴ M, or 10’² M-10’³ M).

In some embodiments, the extracellular CD28-binding domain of the presently described membrane -bound polypeptides has a capacity to bind CD28 that is lower (e.g., up to 1%, up to 2%, up to 3%, up to 4%, up to 5%, up to 6%, up to 7%, up to 8%, up to 9%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, or up to 50 % capacity; or by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90% or more lower; or at least 2 times, 3 times, 4, times, 5 times, 10 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 times, 140 times, or 150 times or more less) than the binding capacity to bind CD28 than that of a B7 costimulatory ligand or the CD28-binding domain of a B7 costimulatory ligand (e.g., having SEQ ID NO: 34 or 35). Such a reduced binding capacity, or affinity, to CD28, is helpful to reduce the avidity of the polypeptide as expressed on the target cell (e.g., a therapeutic cell) so that the therapeutic cell does not aggregate with CD28- expressing host cells outside the context of recognition for destruction by a host T cell. In some embodiments, an extracellular binding domain binds to CD28 with a dissociation constant (KD) of at least 10’⁴ M (e.g., at least IO^-4 M, at least 10’⁵ M, at least 10’⁶ M, at least IO’⁷ M; at least 10’⁸ M; at least 10’⁹ M; at least 10-10 M; at least 10’¹¹ M; at least 10’¹² M; or at least 10’¹³ M). In some embodiments, an extracellular binding domain binds to CD28 with a dissociation constant (KD) of at most 10’⁶ M (e.g., at least 10’⁶ M, at least 10’⁵ M, at least 10’⁴ M, at least 10’³ M; at least 10’² M; at least 10’¹ M; and at least 10° M. In some embodiments, an extracellular binding domain binds to CD28 with a dissociation constant (KD) of 10° M-10’⁶ M (e.g., 10° M-10’⁶ M, 10° M-10’⁵ M, 10° M-10"⁴ M, 10° M-10’³ M, 10’¹ M-10’⁶ M, IO’¹ M-10’⁵ M, IO’¹ M-10^-4 M, IO’¹ M-10’³ M, 10’² M-10’⁶ M, 10’² M-10’⁵ M, 10’² M-10’⁴ M, or 10’² M-10’³ M).

In some embodiments, a CD2-binding domain of any of the membrane-bound polypeptides disclosed herein comprises one or more (e.g., 1, 2, 3, 4 ,5, 6, 7, 8, 9, 10 or more) amino acid substitutions relative to of a CD2-binding domain of LFA-3 (e.g., as set forth in SEQ ID NO: 2). In some embodiments, one or more amino acid substitutions relative to of a CD2-binding domain of LFA-3 reduces the binding capacity, or affinity, of the domain to CD2. In some embodiments, one or more amino acid substitutions in a CD2-binding domain of LFA-3 (e.g., as set forth in SEQ ID NO: 2) are substitutions of amino acids in the C, C’, or G strands. See e.g., Arulanandam et al. (J Exp Med, 1994; 180:1861). In some embodiments, the one or more amino acid substitutions relative to SEQ ID NO: 2 occur at one or more amino acid positions selected from the group consisting of: E25, K29, K32, D33, E37 and R44 of SEQ ID NO: 2. In some embodiments, the substituted amino acid is a nonpolar amino acid (e.g., A, G, I, L, M, F, P, W, or V). In some embodiments, the substituted amino acid is a neutral amino acid (e.g., A, N, C, Q, G, I, L, M, F, P, S, T, W, Y, or V). In some embodiments, the substituted amino acid is an aliphatic amino acid (A, G, I, L, or V). In some embodiments, the substituted amino acid is an aliphatic, nonpolar and/or neutral amino acid. In some embodiments, the substituted amino acid is A. In some embodiments, the one or more amino acid substitutions relative to SEQ ID NO: 2 are selected from the group consisting of: E25A, K29A, K32A, D33A, E37A and R44A relative to SEQ ID NO: 2. In some embodiments, the CD2-binding domain comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of SEQ ID NOs: 2-8, and 48. In some embodiments, the CD28-binding domains comprises one or more sequences at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of SEQ ID NOs: 2-8, and 48.

In some embodiments, a CD28-binding domain of any of the membrane-bound polypeptides disclosed herein comprises one or more (e.g., 1, 2, 3, 4 ,5, 6, 7, 8, 9, 10 or more) amino acid substitutions relative to of a CD28-binding domain of a B7 costimulatory ligand (e.g., as set forth in SEQ ID NOs: 34 and 35). In some embodiments, one or more amino acid substitutions relative to of a CD28-binding domain of a B7 costimulatory ligand reduces the binding capacity, or affinity, of the domain to CD28.

In some embodiments, the CD28-binding domain comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of SEQ ID NOs: 36-39. In some embodiments, the CD28-binding domains comprises one or more sequences at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of SEQ ID NOs: 36-39.

In some embodiments, the extracellular binding domain comprises any one of SEQ ID NOs: 3-8, 34-39, and 48. In some embodiments, the extracellular binding domain comprises an amino acid sequence that is at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%) identical to any one of SEQ ID NOs: 3-8, 34-39, and 48. In some embodiments, the extracellular binding domain comprises one or more (e.g., two or three) domains comprised in any one of SEQ ID NOs: 3-8, 34-39, and 48. In some embodiments, the extracellular binding domain comprises one or more (e.g., two or three) domains as set forth in any one of SEQ ID NOs: 3-8, 34-39, and 48. In some embodiments, the extracellular binding domain comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of the sequences identified in Table 1.

In some embodiments, the extracellular binding domain comprises a signal peptide. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to SEQ ID NO: 33. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NOs: 33, 42, or 43. In some embodiments, the signal peptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NOs: 33, 42, or 43. In some embodiments, the signal peptide comprises SEQ ID NOs: 33, 42, or 43.

In some embodiments, an extracellular portion of a membrane-bound polypeptide as disclosed herein (e.g., comprising an extracellular SMAC-biding domain and elongation domain) is at least 30 amino acids long (e.g., at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids, at least 110 amino acids, at least 120 amino acids long, at least 150 amino acids long, at least 200 amino acids long, at least 250 amino acids long, at least 300 amino acids long, or at least 350 amino acids long or longer). In some embodiments, an extracellular binding domain is at most 400 amino acids long (e.g., at most 400 amino acids, at most 350 amino acids, at most 300 amino acids, at most 250 amino acids, or at most 200 amino acids long). In some embodiments, an extracellular binding domain (e.g., an extracellular CD2-binding domain or an extracellular CD28-binding domain,) is 10-400 amino acids long (e.g., 10-400, 50-200, 50-150, 70-150, 80-120, 80-100, 100-200, 100-400, 200-250, 250-300, or 200-300 amino acids long). In some embodiments, the extracellular binding domain is at least 1 nm (e.g., at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 10 nm, at least 11 nm, at least 12 nm, at least 13 nm, at least 14 nm, at least 15 nm, at least 16 nm, at least 17 nm, at least 18 nm, at least 19 nm, at least 20 nm, at least 21 nm, at least 22 nm, at least 23 nm, at least 24 nm, at least 25 nm, at least 28 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, or more than 50 nm) away from the transmembrane domain when expressed on a cell surface. In some embodiments, the extracellular binding domain is at least 1-60 nm (e.g., 1-10, 5-40, 10-60, 10-50, 10-40, 10-30, 10-25, 12-24, 15-25, 15-30, 5-20, 15-20, 25-30, 1-15, 1-20, 1-30, 5-60, 20-30, 20-40, 20-50, 20-60, 30-40, 30-50, 30-60, 25-50, 25-40, 28-50, 40-50, 50-60 or 40-60 nm) away from the transmembrane domain when expressed on a cell surface.

Extracellular elongation domain

An extracellular elongation domain (e.g., stalk or spacer) of a membrane-bound polypeptide as provided herein acts as a spacer between a host immune cell expressing a SMAC member and the cell (e.g., a therapeutic cell) on which it is expressed. An extracellular elongation domain may be located immediately adjacent to the extracellular binding domain. In some embodiments, the extracellular elongation domain comprises a human sequence. In some embodiments, the extracellular SMAC-binding domain and the extracellular elongation domain are connected with a linker.

The space between a host cell and a cell in which a membrane-bound polypeptide as provided herein is expressed, as provided by the extracellular elongation domain, may be at least 5 nm (e.g., at least 5 nm , at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 28 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm or more than 50 nm). In some embodiments, the space between a host cell and a cell in which a membrane-bound polypeptide as provided herein is expressed, as provided by the extracellular elongation domain, is at least 1-60 nm (e.g., 1-10, 5-40, 10-60, 10-50, 10-40, 10-30, 10-25, 12-24, 15-25, 15-30, 5-20, 15-20, 25-30, 1-15, 1-20, 1-30, 5-60, 20-30, 20-40, 20-50, 20-60, 30-40, 30-50, 30-60, 28-50, 40-50, 50-60 or 40-60 nm). In some embodiments, space between a host cell and a cell in which a membrane-bound polypeptide as provided herein is expressed, as provided by the extracellular elongation domain, is small enough that cells do not aggregate on account of glycocalyx inhibition (e.g., at most 100 ,at most 80, at most 70, at most 60, at most 50, at most 40, at most 30, and at most 25 nm). The extracellular elongation domain provides rigidity to the membrane-bound polypeptide of the present disclosure. In some embodiments, the rigidity is attributed to a lack of flexible domain between the transmembrane domain and extracellular elongation domain. In some embodiments, the rigidity is attributed to the presence of one or more rigid protein modules within the elongation domain.

In some embodiments, an extracellular elongation domain of the membrane-bound polypeptide comprises at least one rigid protein module. In some embodiments, the extracellular elongation domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 rigid protein modules. In some embodiments, the extracellular elongation domain comprises two or more rigid protein modules with the same amino acid sequence. In some embodiments, the extracellular elongation domain comprises two or more rigid protein modules with different amino acid sequences. In some embodiments, one or more rigid protein modules are derived from a human protein. In some embodiments, each of the rigid protein modules are derived from a human protein. In some embodiments, the entire extracellular elongation domain is human.

In some embodiments, a “rigid protein module” refers to a domain having a secondary or tertiary structure that is common to at least two different conformations of a protein comprising the rigid protein module, such that interaction with another molecule (e.g., a ligand) does not result in change in conformation (e.g., does not result in a substantial change in conformation). In some embodiments, rigid protein modules exhibit relatively stiff structures, e.g., by adopting a-helical structures or by containing multiple Proline residues.

The length of the extracellular elongation domain of a membrane-bound polypeptide as described herein can be adjusted, for example by varying the number and size of the rigid protein modules it comprises, to achieve a distance between the extracellular binding domain and the membrane of the cell expressing the membrane-bound polypeptide, such that the membrane-bound polypeptide to CD2 or CD28, expressed on T cells at the time of recognition of the therapeutic cell as a non-self cell, while reducing their binding to CD2 or CD28, outside the context of an immune reaction, thereby avoiding unnecessary cell aggregation and loss of therapeutic cells.

In some embodiments, a rigid protein module is an Ig-like domain of an extracellular protein, e.g., CD22. In some embodiments, the extracellular elongation domain comprises a human sequence. Table 1 provides example sequences comprising 1, 2, 3, 4, or 5 Ig-like domains of CD22, any of which sequences can be incorporated into an elongation domain. In some embodiments, a rigid protein module comprises Cl, C2, C3, C4, C5 or C6 Ig-like domains of CD22 or functional fragments thereof. A functional fragment of a rigid protein domain is a fragment that comprises some fraction (e.g., 90%) of the consecutive amino acids that constitute the domain of which the fragment is a part, and which retains at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5%) rigidity of the Ig-like domain.

In some embodiments, an elongation domain comprises any number and combination of CD22 Ig-like Cl, C2, C3, C4, C5, and C6 domains (UniProt Accession No. P20273). For example, an elongation domain comprises two rigid protein modules each comprising an Ig- like domain of CD22 that may be the same (e.g., two Cl domains) or different (e.g., C5 and C6, or C5 and C4). In some embodiments, the elongation domain comprises three rigid protein modules each comprising an Ig-like domain of CD22, and may be the same or different (e.g., having C4, C5, and C6, or having C6, C6, and C4). In some embodiments, the elongation domain comprises four rigid protein modules each comprising an Ig-like domain of CD22, that may be the same or different (e.g., having C3, C4, C5, and C6; or having C3, C3, C4, and C5). In some embodiments, the elongation domain comprises five rigid protein modules each comprising an Ig-like domain of CD22, and may be the same or different (e.g., having C2, C3, C4, C5, and C6; or having C2, C2, C2, C5, and C6).

In some embodiments, an extracellular elongation domain comprises a first rigid protein module and a second rigid protein module, wherein the first rigid protein module is N-term relative to the second rigid protein module, and wherein the first rigid protein module comprises a sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identical to the C6 Ig-like domain of CD22 as set forth in SEQ ID NO: 29, and the second rigid protein module comprises a sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identical to the C5 Ig-like domain of CD22 as set forth in SEQ ID NO: 28. In some embodiments, an extracellular elongation domain comprises a first rigid protein module and a second rigid protein module, wherein the first rigid protein module is N-term relative to the second rigid protein module, and wherein the first rigid protein module comprises a sequence that is identical to the C6 Ig-like domain of CD22 as set forth in SEQ ID NO: 29, and the second rigid protein module comprises a sequence that is identical to the C5 Ig-like domain of CD22 as set forth in SEQ ID NO: 28.

In some embodiments, an extracellular elongation domain further comprises a third rigid protein module that is C-term relative to the second rigid protein module, wherein the third rigid protein module comprises a sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identical to the C4 Ig-like domain of CD22 as set forth in SEQ ID NO: 27. In some embodiments, an extracellular elongation domain further comprises a third rigid protein module that is C-term relative to the second rigid protein module, wherein the third rigid protein module comprises a sequence that is identical to the C4 Ig-like domain of CD22 as set forth in SEQ ID NO: 27.

In some embodiments, an extracellular elongation domain further comprises a fourth rigid protein module that is C-term relative to the third rigid protein module, wherein the fourth rigid protein module comprises a sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identical to the C3 Ig-like domain of CD22 as set forth in SEQ ID NO: 26. In some embodiments, an extracellular elongation domain further comprises a fourth rigid protein module that is C-term relative to the third rigid protein module, wherein the fourth rigid protein module comprises a sequence that is identical to the C3 Ig-like domain of CD22 as set forth in SEQ ID NO: 26.

In some embodiments, an extracellular elongation domain further comprises a fifth rigid protein module that is C-term relative to the fourth rigid protein module, wherein the fifth rigid protein module comprises a sequence that is at least 85% (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%) identical to the C2 Ig-like domain of CD22 as set forth in SEQ ID NO: 25. In some embodiments, an extracellular elongation domain further comprises a fifth rigid protein module that is C-term relative to the fourth rigid protein module, wherein the fifth rigid protein module comprises a sequence that is identical to the C2 Ig-like domain of CD22 as set forth in SEQ ID NO: 25.

In some embodiments, the extracellular elongation domain comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of the sequences identified in Table 1.

In some embodiments, the extracellular elongation domain comprises one or more domains of a CD45 protein (e.g., CD45RO, or CD45RABC), or a functional fragment thereof.

In some embodiments, an extracellular elongation domain of a membrane-bound polypeptide as provided herein is at least 100 amino acids long (e.g., at least 100 amino acids, at least 110 amino acids, at least 120 amino acids long, at least 150 amino acids long, at least 160 amino acids long, at least 170 amino acids long, at least 180 amino acids long, at least 190 amino acids long, at least 200 amino acids long, at least 250 amino acids long, at least 300 amino acids long, at least 350 amino acids long, at least 400 amino acids long, at least 450 amino acids long, at least 500 amino acids long, at least 525 amino acids long, at least 550 amino acids long, at least 575 amino acids long, at least 600 amino acids long, or at least 650 amino acids long). In some embodiments, an extracellular elongation domain is at most 900 amino acids long (e.g., at most 900 amino acids, at most 800 amino acids, at most 700 amino acids, at most 600 amino acids, at most 500 amino acids long, at most 450 amino acids long, at most 400 amino acids long, at most 300 amino acids long, at most 200 amino acids long). In some embodiments, an extracellular elongation domain is 100-1,000 amino acids long (e.g., 100-1000, 200-1000, 300-1000, 400-1000, 200-1000, 300-1000, 400-1000, 200- 500, 200-300, 200-400, 300-500, 300-600, 100-800, 200-800, 300-800, 400-800 amino acids long). In some embodiments, an extracellular elongation domain is 200-800 amino acids long (e.g., 200-800, 200-600, 250-550, 300-500, 350-500, 300-400, 400-500, 400-600, 300- 800, 400-800, 400-600, or 300-700 amino acids long).

In some embodiments, an extracellular elongation domain is at least 10 nm (e.g., at least 10 nm, at least 12 nm, at least 15 nm, at least 17.5 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 55 nm or at least 60 nm or more) in length. In some embodiments, each of the one or more rigid protein modules is at least 1 nm (e.g., at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 10 nm, or at least 20 nm or more) in length. In some embodiments, the extracellular elongation domain does not comprise a domain of CD45, CD48, CD58, CD28, or CD2.

In some embodiments, the length of the extracellular portion of the membrane-bound polypeptide (e.g., comprising an extracellular CD2-binding domain and an extracellular elongation domain, comprising an extracellular CD2-binding domain and an extracellular elongation domain with a linker, comprising an extracellular CD28-binding domain and an extracellular elongation domain, or comprising an extracellular CD28-binding domain and an extracellular elongation domain with a linker) is at least 10 nm (e.g., at least 10 nm, at least 12 nm, at least 15 nm, at least 17.5 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 55 nm, at least 60, at least 70, at least 80, at least 90, or at least 100 nm or more). In some embodiments, the length of the extracellular portion of the membrane -bound polypeptide (e.g., comprising an extracellular CD2-binding domain and an extracellular elongation domain, comprising an extracellular CD2-binding domain and an extracellular elongation domain with a linker, comprising an extracellular CD28-binding domain and an extracellular elongation domain, or comprising an extracellular CD28-binding domain and an extracellular elongation domain with a linker) is 10-100 nm (e.g., 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-25, 12-24, 15-25, 15-30, 5-20, 15-20, 25-30, 1-15, 1-20, 1-30, 5-60, 20-30, 25-30, 25-35, 25-40, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-100, 30-40, 30-50, 30-60, 28-50, 30-70, 30-80, 30-90, 30-100, 40-50, 40-70, 40-80, 40-90, 40-100, 50-60, 50-70, 50-80, 50-90, 50-100, 60-70, 60- 80, 60-90, 60-100, 70-80, 70-90, 70-100, 80-90, or 90-100 nm).

Transmembrane domain

As used herein, a “transmembrane domain” refers to a domain of a membrane-bound polypeptide as provided herein that is embedded in the phospholipid bilayer of a cell expressing the membrane -bound polypeptide.

In some embodiments, the transmembrane domain and the extracellular elongation domain are derived from the same protein. In some embodiments, having a transmembrane domain and an extracellular elongation domain derived from the same protein provides rigidity to the membrane-bound polypeptide.

In some embodiments, the transmembrane domain comprises human sequence. In some embodiments, the transmembrane domain is a portion of a human membrane-bound protein (e.g., CD22, CD80 or CD86). In some embodiments, the extracellular elongation domain comprises one or more domains of a CD45 protein (e.g., CD45RO, or CD45RABC), or a functional fragment thereof. In some embodiments, the transmembrane domain does not comprise a transmembrane domain of CD45, CD48, CD58, CD2, or CD28.

Table 1 provides examples of transmembrane domains as comprised in the membrane-bound polypeptides of the present application. In some embodiments a membrane-bound polypeptide as provided herein has a transmembrane domain comprising an amino acid sequence that is at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100%) identical to any one the transmembrane domains disclosed in Table 1.

In some embodiments, a transmembrane domain is at least 5 amino acids long (e.g., at least 5, at least 10, at least 12, at least 15, at least 20, at least 25, or at least 30 amino acids long). In some embodiments, a transmembrane domain is at most 100 amino acids long (e.g., at most 100, at most 80, or at most 50 amino acids long). In some embodiments, a transmembrane domain is 10-100 amino acids long (e.g., 10-100, 10-80, 10-70, 10-60, 10-50, 10-40, 20-40, 20-30, 25-35, 20-40, 25-50, 30-50, 30-35, 30-40, 40-50, 40-60, 40-80, or 40- 100 amino acids long). In some embodiments, a transmembrane domain is 2-20 nm (e.g., 2- 20, 2-15, 2-10, 4-10, 4-15, 4-20, 5-15, 5-10, 7-10, 7-15 or 7.5-12.5 nm) long. In some embodiments, a transmembrane domain is 5-10 nm long, for example, 6 nm, 7 nm, 8 nm, or 9 nm. Linker

As used herein, a linker connects two domains of a membrane-bound polypeptide as disclosed herein. In some embodiments, a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some embodiments, the linker is flexible, such as those comprising one or more glycines. In some embodiments, a linker comprises a series of glycine and/or serine repeats. In some embodiments, the linker comprises one or more glycine- serine repeats selected from the group consisting of GGS, GGGS (SEQ ID NO: 1), GGGGS (SEQ ID NO: 44), GSS, GSSSSS (SEQ ID NO: 45), GGGGSS (SEQ ID NO: 46), or GGSSSS (SEQ ID NO: 47). In some embodiments, a linker comprises an amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the linker is a CD8 linker. In some embodiments, a linker is absent between two domains of the membrane-bound polypeptide as disclosed herein. In some embodiments, a lack of linker provides rigidity to the polypeptide.

In some embodiments, the extracellular binding domain is linked to the extracellular elongation domain by a linker. In some embodiments, the linker comprises SEQ ID NO: 1. In some embodiments, the linker has a sequence identical to SEQ ID NO: 1.

In some embodiments, the extracellular binding domain is linked to the extracellular elongation domain by a CD8 linker.

In some embodiments, a linker comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of the linker sequences identified in Table 1.

Intracellular domain

In some embodiments, a membrane-bound polypeptide as disclosed herein further comprises an intracellular domain. As used herein, “intracellular domain” refers to a domain of the membrane-bound polypeptide that is present in the cytoplasm of the cell in which the membrane-bound polypeptide is expressed. In some embodiments, the intracellular domain is connected to the transmembrane domain by a linker. In some embodiments, the intracellular domain is capable of associating, or co-clustering with, MHC molecules.

In some embodiments, an intracellular domain of a membrane-bound polypeptide provided herein comprises one or more intracellular domains of LFA-3 (including CD48 or CD58) or one or more intracellular domains or a B7 costimulatory ligand (e.g., CD80 or CD86). In some embodiments, the intracellular domain comprises a human sequence. In some embodiments, the intracellular domain of a membrane-bound polypeptide provided herein is an intracellular domain of CD22. In some embodiments, the intracellular domain and transmembrane domain of a membrane-bound polypeptide provided herein are from the same protein. Table 1 provides examples of intracellular domains that can be comprised in any one of the membrane -bound polypeptides of the present disclosure. In some embodiments a membrane-bound polypeptide as provided herein has an intracellular domain comprising an amino acid sequence that is at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, and 100%) identical to any one of the intracellular domains disclosed in Table 1.

Stalk

In some embodiments, a membrane-bound polypeptide of the present disclosure comprises an extracellular SMAC -binding domain (e.g., an extracellular CD2-binding domain or an extracellular CD28-binding domain) comprising a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 2-8 and 34-39; and a stalk (e.g., Ig-like domains and transmembrane domain from CD22) comprising an extracellular domain, a transmembrane domain, and optionally an intracellular domain sequence. In some embodiments, a stalk is that part of the polypeptide that is not the SMAC-binding domain (e.g., an extracellular CD2-binding domain or an extracellular CD28-binding domain). In some embodiments, a stalk comprises an extracellular elongation domain, transmembrane domain and/or an intracellular domain as disclosed herein. In some embodiments, the stalk comprises a human sequence. In some embodiments, the stalk comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of the sequences identified in Table 1.

In some embodiments, the stalk comprises one or more domains of a CD45 protein (e.g., CD45RO, or CD45RABC), or a functional fragment thereof.

Methods of measuring rigidity of a polypeptide, such as any of the membrane-bound polypeptides provided herein, or any domain(s) thereof, are known in the art. In some embodiments, sedimentation, gel filtration, electron microscopy and rotary shadow electron microscopy can be used to evaluate the size and shape of proteins. See, e.g., Erickson (Shulin Li (ed.), Biological Procedures Online, Volume 11, Number 1) and Chang et al. Nat Immunol. 2016. 17(5):574-582. In some embodiments, X-ray crystallography or NMR spectroscopy or cryo-electron microscopy or cryo-tomo election microscopy is used to measure shape, size and/or dimensions of a protein. In some embodiments, rigidity is measured by calculating the rotational freedom in any domain pair in a protein. In some embodiments, rigidity is measured by calculating the rotational freedom between each domain pair in a protein. Further, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) can be used to measure how upright a protein is relative to the cell surface. In some embodiments, the rotational freedom of extracellular elongation domains present in a membrane-bound polypeptide as provided herein is 15° or less, 10° or less, 9° or less, 8° or less, 7° or less, 6° or less, 5° or less, 4° or less, 3° or less, 2° or less, or 1° or less. In some embodiments, the rigidity of extracellular elongation domains present in a membranebound polypeptide as provided herein is 15° or less, 10° or less, 9° or less, 8° or less, 7° or less, 6° or less, 5° or less, 4° or less, 3° or less, 2° or less, or 1° or less.

In some aspects, the disclosure provides a membrane-bound polypeptide comprising an extracellular binding domain comprising a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of SEQ ID NOs: 2- 8, and 34-39; and a CD22 stalk comprising an extracellular domain, a transmembrane domain, and optionally an intracellular domain sequence. In some embodiments, the CD22 stalk comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of SEQ ID NOs: 9-12. In some embodiments, the extracellular binding domain is linked to the CD22 stalk via a linker comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the linker has a sequence identical to SEQ ID NO: 1.

Exemplary constructs of membrane-bound polypeptides

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 1).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain, an extracellular elongation domain comprising the C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 2).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain, an extracellular elongation domain comprising the C4, C5 and C6 Ig- like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 3). In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain, an extracellular elongation domain comprising the C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 4).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain comprising an E25A substitution relative to SEQ ID NO: 2, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 5).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain comprising a K29A substitution relative to SEQ ID NO: 2, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 6).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain comprising a K32A substitution relative to SEQ ID NO: 2, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 7).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain comprising a D33A substitution relative to SEQ ID NO: 2, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 8).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain comprising an E37A substitution relative to SEQ ID NO: 2, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 9).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain comprising an R44A substitution relative to SEQ ID NO: 2, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 10).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain comprising a K32A substitution relative to SEQ ID NO: 2, an extracellular elongation domain comprising the C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 11).

In some embodiments, a membrane-bound polypeptide comprises a CD80 extracellular domain, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 12).

In some embodiments, a membrane-bound polypeptide comprises a CD86 extracellular domain, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 13).

In some embodiments, a membrane-bound polypeptide comprises either one CD58, two CD58, or a scFv (e.g., VH/VL) extracellular domain; an extracellular elongation domain comprising either the C2, C3, C4, C5 and C6 Ig-like domains of CD22, the CD45RO domain, or the CD45RABC domain; and a transmembrane domain, and optionally an intracellular domain (FIG. 14, left).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 14, middle).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain, an extracellular elongation domain comprising the CD45RO domain, and a transmembrane domain, and optionally an intracellular domain (FIG. 14, right).

In some embodiments, a membrane-bound polypeptide comprises a CD58 extracellular domain, an extracellular elongation domain comprising the CD45RABC domain, and a transmembrane domain, and optionally an intracellular domain (FIG. 14).

In some embodiments, a membrane-bound polypeptide comprises two CD58 extracellular domains, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 14).

In some embodiments, a membrane-bound polypeptide comprises two CD58, extracellular domains, an extracellular elongation domain comprising the CD45RO domain, and a transmembrane domain, and optionally an intracellular domain (FIG. 14).

In some embodiments, a membrane-bound polypeptide comprises two CD58 extracellular domains, an extracellular elongation domain comprising the CD45RABC domain, and a transmembrane domain, and optionally an intracellular domain (FIG. 14).

In some embodiments, a membrane-bound polypeptide comprises a scFv (e.g., VH/VL) extracellular domain, an extracellular elongation domain comprising the C2, C3, C4, C5 and C6 Ig-like domains of CD22, and a transmembrane domain, and optionally an intracellular domain (FIG. 14).

In some embodiments, a membrane-bound polypeptide comprises a scFv (e.g., VH/VL) extracellular domain, an extracellular elongation domain comprising the CD45RO domain, and a transmembrane domain, and optionally an intracellular domain (FIG. 14).

In some embodiments, a membrane-bound polypeptide comprises a scFv (e.g., VH/VL) extracellular domain, an extracellular elongation domain comprising the CD45RABC domain, and a transmembrane domain, and optionally an intracellular domain (FIG. 14).

In some embodiments, a membrane-bound polypeptide as provided herein further comprises a signal peptide.

In some aspects, the disclosure provides a membrane-bound polypeptide comprising a CD2-binding domain or CD28-binding domain, wherein the membrane-bound polypeptide comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of SEQ ID NOs: 13-22, 40, 41, and 48.

In some embodiments, a membrane-bound polypeptide as provided herein comprises a sequence that is at least 85% (e.g., at least 90%, at least 95%, at least 98%, or at least 99%, or 100%) identical to any one of the sequences identified in Table 1.

Nucleic acids encoding membrane-bound polypeptides (e.g., decoy receptors), and vectors comprising them

In some aspects, the disclosure provides a nucleic acid comprising a sequence encoding any one of the membrane-bound polypeptides as provided herein.

In some embodiments, a nucleic acid encoding any one of the membrane-bound polypeptides as provided herein is comprised on a vector. A vector is a vehicle for delivering nucleic acid into a cell (e.g., an in vitro cell or in vivo cell). In some embodiments, a vector as provided herein further comprises a control element that is operably linked to the nucleic acid sequence encoding any one of the membrane-bound polypeptides as provided herein. Non-limiting examples of a control element include promoters, insulators, silencers, response elements, introns, enhancers, initiation sites, termination signals, and poly(A) tails. Any combination of such control sequences is contemplated herein (e.g., a promoter and an enhancer). A promoter may be, for example, a constitutive promoter, a controllable promoter, a tissue- specific promoter, an inducible promoter, or a synthetic promoter. An inducible promoter that can control the level of expression of a membrane-bound polypeptide as described herein is useful for to control the avidity of a cell that expresses the polypeptide to cells expressing a SMAC (e.g., CD2 or CD28).

A vector as described herein may be a DNA or RNA vector. A vector may be linear or circular. A vector may be viral or non-viral. A viral vector may be integrating (e.g., a lentiviral vector, or retroviral vector) or a non-integrating vector (e.g., AAV, adenovirus, HSV, or baculovirus). A non-limiting example of a non-viral vector is a plasmid. In some embodiments a vector is an artificial delivery formulation, e.g., a lipid nanoparticle, that incorporates the nucleic acid.

Method of producing a therapeutic cell capable of evading host immune system

In some aspects, the disclosure provides a method for producing a therapeutic cell capable of evading a host immune system, the method comprising inserting (e.g., introducing) into the therapeutic cell a nucleic acid as provided herein, contacting the cell with a vector as provided herein, or expressing in the cell a membrane-bound polypeptide as provided herein. In some embodiments, a formulation comprising a nucleic acid or vector as provided herein is contacted with the therapeutic cell in culture. In some embodiments, the cell is an allogeneic cell. In some embodiment, the cell is an autologous cell.

In some embodiments, the disclosure provides methods for producing therapeutic cells that express at least 100 (e.g., at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1,000, at least 5,000, at least 10,000, at least 50,000, at least 100,000, or at least 500,000) membrane -bound polypeptides of the present disclosure on their surface. The level of expression is a factor that affect the avidity of membrane -bound polypeptides expressed on a cell (e.g., a therapeutic cell) to cells that express a SMAC (e.g., CD2 or CD28). In some embodiments, the therapeutic cells express 10²-10⁷ (e.g., 100-1,000, 10³-10⁴, 10⁴-10⁵, 10⁵-10⁶, 10⁵-10⁷, 10²-10⁴, 10²-10⁵, 10²-10⁶, 10³-10⁴, 10³-10⁵, 10³-10⁶, 10³- 10⁷, 10⁴-10⁶, 10⁴-10⁷, and 10⁶-10⁷) membrane-bound polypeptides of the present disclosure on their surface. In some embodiments, the therapeutic cells express no more than 10⁷ cells (e.g., no more than 10⁷, no more than 10⁶, no more than 10⁵, no more than 10⁴, no more than 10³) membrane-bound polypeptides of the present disclosure on their surface. Examples of methods for introducing a vector or nucleic acid as described herein in a cell include, but are not limited to, transfection, transduction, transformation, and infection. In some embodiments, a physical method such as electroporation, sonoporation, magnetofection, direct micro injection, biolistic particle delivery, or laser-based transfection is used. In some embodiments, transfection of cell with nucleic acid is transient. In some embodiments, transfection of cell with nucleic acid is stable.

Cells expressing membrane-bound polypeptides (e.g., decoy receptors)

In some aspects, the disclosure provides a cell expressing a membrane-bound polypeptide as provided herein, or comprising a nucleic acid or a vector as provided herein. In some embodiments, a cell expressing any one of the membrane-bound polypeptide as provided herein is a therapeutic cell, intended to be administered to a subject (e.g., to treat a disease or condition in the subject). In some aspects, the present disclosure provides a therapeutic cell capable of evading host immune system.

In some embodiments, a cell as provided herein expresses at least 100 (e.g., at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1,000, at least 5,000, at least 10,000, at least 50,000, at least 100,000, or at least 500,000) membrane-bound polypeptides of the present disclosure on their surface. The level of expression is a factor that affect the avidity of membrane-bound polypeptides expressed on a cell (e.g., a therapeutic cell) to cells that express a SMAC (e.g., CD2 or CD28). In some embodiments, the therapeutic cells express 10²-10⁷ (e.g., 100-1,000, 10³-10⁴, 10⁴-10⁵, 10⁵-10⁶, 10⁵-10⁷, 10²- 10⁴, 10²-10⁵, 10²-10⁶, 10³-10⁴, 10³-10⁵, 10³-10⁶, 10³-10⁷, 10⁴-10⁶, 10⁴-10⁷, and 10⁶-10⁷) membrane-bound polypeptides of the present disclosure on their surface. In some embodiments, the therapeutic cells express no more than 10⁷ cells (e.g., no more than 10⁷, no more than 10⁶, no more than 10⁵, no more than 10⁴, no more than 10³) membrane-bound polypeptides of the present disclosure on their surface.

A therapeutic cell may be one of many cells cultured under certain conditions, or part of an organ that is harvested, part of an organoid, or an organism. In some embodiments, a cell disclosed herein is a eukaryotic cell (derived from a eukaryotic organism). In some embodiments, a eukaryotic cell is derived from ectoderm, endoderm, or mesoderm.

In some embodiments, a therapeutic cell is a mammalian cell. In some embodiments, a therapeutic cell is a human cell. In some embodiments, a therapeutic cell is an allogeneic cell. In some embodiments, a therapeutic cell as disclosed herein is a stem cell (e.g., an induced pluripotent stem cell). In some embodiments, a disclosed herein is immortalized (e.g., HEK293 cell, A549 cell, HeLa cell, Jurkat cell, 3T3 cell, or Vero cell).

In some embodiments, a therapeutic cell is an immune cell. Non-limited examples of immune cells include granulocytes, mast cells, monocytes, neutrophils, dendritic cells, NK cells, or adaptive cells like B cells and T cells. T cells may be cytotoxic T cells, helper T cells or regulatory T cells. In some embodiments, a cell is a lymphocyte (e.g., a NK1.1+ cell, CD3+ cell, CD4+ cell, or CD8+ cell). In some embodiments, a therapeutic cell (e.g., allogeneic cell and autologous cell) is a T cell, a precursor T cell, or a hematopoietic stem cell. In some embodiments, the cell is a CD4+ T cell (e.g., a FOXP3-CD4+ T cell or a FOXP3+CD4+ T cell) or a CD8+ T cell (e.g., a FOXP3-CD8+ T cell or a FOXP3+CD8+ T cell). In some embodiments, the cell is an NK-T cell (e.g., a FOXP3- NK-T cell or a FOXP3+ NK-T cell). In some embodiments, the cell is a regulatory B (Breg) cell (e.g., a FOXP3- B cell or a FOXP3+ B cell). In some embodiments, the cell is a CD25- T cell. In some embodiments, the cell is a regulatory T (Treg) cell. Non-limiting examples of Treg cells are Tri, Th3, CD8+CD28-, and Qa-1 restricted T cells. In some embodiments, the Treg cell is a FOXP3+ Treg cell. In some embodiments, the Treg cell expresses CTLA-4, LAG-3, CD25, CD39, neuropilin- 1, galectin-1, and/or IL-2Ra on its surface.

In some embodiments, a therapeutic cell is an engineered cell, e.g., modified phenotypically by genetic engineering (e.g., genome modification or gene expression). Any of the aforementioned cells may be an engineered cell. Non-limiting examples of engineered therapeutic cells contemplated herein include engineered T cells (e.g., engineered cytotoxic T cell and engineered regulatory T cells), engineered regulatory B cells, or engineered NK cells. Non-immune therapeutic cells may also be engineered.

In some embodiments, a therapeutic cell further expresses a chimeric antigen receptor (CAR), or a T-cell receptor. In some embodiments, a therapeutic cell is an engineered cell further expressing a chimeric antigen receptor, or a T-cell receptor. In some embodiments, a therapeutic cell is a T cell further expressing a chimeric antigen receptor, or a T-cell receptor. In some embodiments, a therapeutic cell is an engineered T cell e.g., engineered cytotoxic T cell and engineered regulatory T cells) further expressing a chimeric antigen receptor, or a T- cell receptor. A CAR or TCR may bind to a therapeutic target (for example to direct a therapeutic cell (e.g., an engineered Treg cell to a target cell or tissue).

In some embodiments, a therapeutic cell is a cell or an organ engineered to reform or repair an organ or part thereof.

In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vivo. In some embodiments, a cell as provided herein is an engineered cell. In some embodiments, an engineered cell is a cell in which one or more genes/loci are manipulated or edited (e.g., to express one or more exogenous genes). In some embodiments, the cell is a human cell. Provided herein are compositions, e.g., pharmaceutical compositions, comprising a multiplicity of any one of the therapeutic cells as disclosed herein. In some embodiments, a pharmaceutical composition of therapeutic cells comprises an isotonic solution. In some embodiments, a pharmaceutical composition of therapeutic cells comprises a stabilizer (e.g., albumin). In some embodiments, a pharmaceutical composition of therapeutic cells comprises 10³-10¹² cells (e.g., 10³-10¹², 10³-10ⁿ, 1O³-1O¹⁰, 10³-10⁹, 10³-10⁸, 10³-10⁷, 10³-10⁶,

10³-10⁵, 10³-10⁴, 10⁴-10¹², 10⁴-10ⁿ, 1O⁴-1O¹⁰, 10⁴-10⁹, 10⁴-10⁸, 10⁴-10⁷, 10⁴-10⁶, 10⁴-10⁵,

10⁵-10¹², 10⁵-10ⁿ, 1O⁵-1O¹⁰, 10⁵-10⁹, 10⁵-10⁸, 10⁵-10⁷, 10⁵-10⁶, 10⁶-10¹², 10⁶-10ⁿ, 1O⁶-1O¹⁰,

10⁶-10⁹, 10⁶-10⁸, 10⁶-10⁷, 10⁷-10¹², 10⁷-10ⁿ, 1O⁷-1O¹⁰, 10⁷-10⁹, 10⁷-10⁸, or any discrete number encompassed therein).

Methods of administering

Provided herein is a method comprising administering to a subject any one of the therapeutic cells capable of evading host immune attack, as disclosed herein, e.g., to treat a disease or condition in the subject. In some embodiments, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful. To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. A therapeutic amount may be comprises 10³-10¹² cells (e.g., 10³-10¹², 10³-10ⁿ, 1O³-1O¹⁰, 10³- 10⁹, 10³-10⁸, 10³-10⁷, 10³-10⁶, 10³-10⁵, 10³-10⁴, 10⁴-10¹², 10⁴-10ⁿ, 1O⁴-1O¹⁰, 10⁴-10⁹, 10⁴-10⁸,

10⁴-10⁷, 10⁴-10⁶, 10⁴-10⁵, 10⁵-10¹², 10⁵-10ⁿ, 1O⁵-1O¹⁰, 10⁵-10⁹, 10⁵-10⁸, 10⁵-10⁷, 10⁵-10⁶,

10⁶-10¹², 10⁶-10ⁿ, 1O⁶-1O¹⁰, 10⁶-10⁹, 10⁶-10⁸, 10⁶-10⁷, 10⁷-10¹², 10⁷-10ⁿ, 1O⁷-1O¹⁰, 10⁷-10⁹,

10⁷-10⁸, or any discrete number encompassed therein), and may be administered to a subject in a single dose, or over multiple doses (e.g., 2, 3, 4, 5, or more than 5) doses, either simultaneously or sequentially.

In some embodiments, the subject is a human. In some embodiments, the subject has or is at risk of developing a disease or condition selected from an autoimmune condition, an allergic condition, and/or an inflammatory condition. In some embodiments, an autoimmune disease or condition is type 1 diabetes mellitus, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, early onset rheumatoid arthritis, ankylosing spondylitis, immune-mediated pregnancy loss, immune-mediated recurrent pregnancy loss, dermatomyositis, psoriatic arthritis, Crohn’s disease, bullous pemphigoid, pemphigus vulgaris, autoimmune hepatitis, psoriasis, Sjogren’s syndrome, or celiac disease. In some embodiments, an allergic condition is allergic asthma, atopic dermatitis, pollen allergy, food allergy, drug hypersensitivity, or contact dermatitis. In some embodiments, an inflammatory condition is pancreatic islet cell transplantation, asthma, steroid-resistant asthma, hepatitis, traumatic brain injury, primary sclerosing cholangitis, primary biliary cholangitis, polymyositis, stroke, Still’s disease, acute respiratory distress syndrome (ARDS), uveitis, inflammatory bowel disease (IBD), ulcerative colitis, graft-versus-host disease (GVHD), tolerance induction for transplantation, transplant rejection, or sepsis. In some embodiments, the subject has or is at risk of developing type 1 diabetes mellitus. In some embodiments, the subject has or is at risk of developing inflammatory bowel disease. In some embodiments, the subject has or is at risk of developing acute respiratory distress syndrome (ARDS).

Formulations comprising cells (e.g., therapeutic cells) as described herein that are administered to a subject comprise a number of therapeutic cells that is effective for the treatment and/or prophylaxis of the specific indication or disease to be treated by the cell therapy. Thus, therapeutically-effective populations therapeutic cells as described herein can be administered to subjects. In some embodiments, formulations are administered that comprise between about IxlO⁴ and about IxlO¹⁰ therapeutic cells. In some embodiments, the formulations comprise between about IxlO⁵ and about IxlO⁹ therapeutic cells, between about 5xl0⁵ and about 5xl0⁸ therapeutic cells, or between about IxlO⁶ to about IxlO⁷ therapeutic cells. In some embodiments, the number of therapeutic cells administered to a subject depends upon the anatomical location, administration route, cell source, patient age and medical history, indication, disease severity, and other factors.

Kits

The compositions comprising nucleic acids, or vectors as described herein may be included in a kit. In some embodiments, the disclosure provides kits comprising media, growth factors, antibodies (e.g., for cell sorting or characterization) and / or plasmids encoding the membrane-bound polypeptides as described herein. Reagents suitable for cell growth and / or differentiation, and for plasmid transfection may be included in the kits provided herein.

In some embodiments, the kit includes reagents or devices for electroporation of cells. The kit may include one or more compositions or reagents that can be combined to produce a composition of any embodiment of the present disclosure. In some embodiments, the components of the kit are stored in an aqueous medium or in a lyophilized form. In some embodiments, the kit comprises at least one container selected from a vial, test tube, flask, bottle, syringe, or other container in which the components of the kits are placed, preferably separated from each other. Where there are multiple components in the kit, the kit also typically includes a second, third, or other additional container in which the additional components of the kits are placed separately. Various combinations of components may be included in the kits provided herein. In some embodiments, kits further comprise cells, nucleic acid constructs, and other reagents. In some embodiments, containers comprised in the kit are tightly closed for commercial use. Such containers may include, without limitation, injection molding or blow molding in which a vial is positioned.

In some aspects, the disclosure provides kits comprising compositions. In some aspects, the disclosure provides kits for the treatment or prevention of a disease. In one embodiment, a kit can include a therapeutic or prophylactic composition containing an effective amount of cells (e.g., therapeutic cells) as described herein. In some cases, a kit can include from about IxlO⁴ cells to about IxlO¹² cells. In some cases a kit can include at least about IxlO⁵ cells, at least about IxlO⁶ cells, at least about IxlO⁷ cells, at least about 4xl0⁷ cells, at least about 5xl0⁷ cells, at least about 6xl0⁷ cells, at least about 6xl0⁷ cells, at least about 8xl0⁷ cells, at least about 9xl0⁷ cells, at least about IxlO⁸ cells, at least about 2xl0⁸ cells, at least about 3xl0⁸ cells, at least about 4xl0⁸ cells, at least about 5xl0⁸ cells, at least about 6xl0⁸ cells, at least about 6xl0⁸ cells, at least about 8xl0⁸ cells, at least about 9xl0⁸ cells, at least about IxlO⁹ cells, at least about 2xl0⁹ cells, at least about 3xl0⁹ cells, at least about 4xl0⁹ cells, at least about 5xl0⁹ cells, at least about 6xl0⁹ cells, at least about 6xl0⁹ cells, at least about 8xl0⁹ cells, at least about 9xl0⁹ cells, at least about IxlO¹⁰ cells, at least about 2xlO¹⁰ cells, at least about 3xlO¹⁰ cells, at least about 4xlO¹⁰ cells, at least about 5xlO¹⁰ cells, at least about 6xlO¹⁰ cells, at least about 6xlO¹⁰ cells, at least about 8xlO¹⁰ cells, at least about 9xlO¹⁰ cells, at least about IxlO¹¹ cells, at least about 2xlOⁿ cells, at least about 3xlOⁿ cells, at least about 4xlOⁿ cells, at least about 5xlOⁿ cells, at least about 6xlOⁿ cells, at least about 6xlOⁿ cells, at least about 8xlOⁿ cells, at least about 9xlOⁿ cells, or at least about IxlO¹² cells. For example, about 5xlO¹⁰ cells may be included in a kit. In another example, a kit may include 3xl0⁶ cells; the cells may be expanded to about 5xlO¹⁰ cells and administered to a subject.

In some embodiments, a kit described herein includes cells (e.g., therapeutic cells) which are allogenic or autologous cells. In some embodiments, a kit described herein includes cells (e.g., therapeutic cells) that comprise a genomic modification. In some embodiments, a kit described herein comprises “off-the-shelf’ cells. In some embodiments, a kit described herein includes cells that may be expanded for clinical use. In some embodiments, a kit described herein contains materials (e.g., components) for research purposes.

In some embodiments, a kit described herein comprises instructions that include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a disease or condition; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

EXAMPLES

Example 1: Effect of CD2-binding membrane-bound polypeptide expression on survival of allogeneic T cells in an in vitro model

PBMCs are obtained from two healthy human subjects with two different HLA serotypes, Subject 1 and Subject 2. T cells from Subject 2 are isolated and exposed to dendritic cells from Subject 1 for 5-7 days to allow stimulation of the T cells from Subject 2 against antigens from Subject 1. T cells obtained from Subject 1 are then transfected with plasmids encoding a sequence selected from SEQ ID NOs: 13-22, or with a control plasmid. CFSE staining is used to evaluate the expansion of cells reactive towards T cells transfected with control plasmids, compared to cells that express a membrane-bound polypeptide as described herein (e.g., SEQ ID NOs: 13-22).

A one-way MLR experiment is then performed by co-culturing stimulated T cells from Subject 2 with T cells from Subject 1. Activation of the stimulated cells is evaluated by diluting CFSE and staining for intracellular IFN-y. Results show that there is a significant reduction in expansion of T cells from Subject 2 when co-cultured with T cells from Subject 1 that express a membrane-bound polypeptide as described herein (e.g., SEQ ID NOs: 13- 22), compared to when co-cultured with T cells from Subject 1 transfected with control plasmids.

Example 2: Effect of CD2-binding membrane-bound polypeptide expression on survival of allogeneic Tregs in a mouse model

Mouse regulatory T cells (Treg cells) obtained from donor mice are grown in culture and transfected by electroporation with plasmids comprising a sequence encoding an amino acid sequence selected from SEQ ID NOs: 13-22 and a sequence encoding a Green Fluorescent Protein (GFP). Control Treg cells obtained from the same mouse donors are transfected with control plasmids encoding only GFP. Plasmid uptake by cells is verified by fluorescence imaging. The transfected Tregs are administered to recipient mice (different from the donor mice) via intramuscular injection. Fluorescence imaging conducted on mouse blood samples immediately after injection, and then 1, 3, 5, and 7 days after injection show a significant decrease over time of the fluorescence signal in the control group compared to the other experimental groups where fluorescence levels are sustained. These results indicate the increased in vivo viability of allogeneic Tregs expressing the membrane-bound polypeptides described in the present disclosure, compared to control allogeneic Tregs.

Example 3: Effect of CD28-binding membrane-bound polypeptide expression on survival of allogeneic T cells in an in vitro model

PBMCs are obtained from two healthy human subjects with two different HLA serotypes, Subject 1 and Subject 2. T cells from Subject 2 are isolated and exposed to dendritic cells from Subject 1 for 5-7 days to allow stimulation of the T cells from Subject 2 against antigens from Subject 1. T cells obtained from Subject 1 are then transfected with plasmids encoding a sequence selected from SEQ ID NO: 40 or 41, or with a control plasmid. CFSE staining is used to evaluate the expansion of cells reactive toward T cells transfected with control plasmids, compared to cells that express a membrane-bound polypeptide as described herein (e.g., SEQ ID NO: 40 or 41).

A one-way MLR experiment is then performed by co-culturing stimulated T cells from Subject 2 with T cells from Subject 1. Activation of the stimulated cells is evaluated by diluting CFSE and staining for intracellular IFN-y. Results show that there is a significant reduction in expansion of T cells from Subject 2 when co-cultured with T cells from Subject 1 that express a membrane-bound polypeptide as described herein (e.g., SEQ ID NO: 40 or 41), compared to when co-cultured with T cells from Subject 1 transfected with control plasmids.

Example 4: Effect of CD28-binding membrane-bound polypeptide expression on survival of allogeneic Tregs in a mouse model

Mouse regulatory T cells (Treg cells) obtained from donor mice are grown in culture and transfected by electroporation with plasmids comprising a sequence encoding an amino acid sequence selected from SEQ ID NO: 40 or 41 and a sequence encoding a Green Fluorescent Protein (GFP). Control Treg cells obtained from the same mouse donors are transfected with control plasmids encoding only GFP. Plasmid uptake by cells is verified by fluorescence imaging. The transfected Tregs are administered to recipient mice (different from the donor mice) via intramuscular injection. Fluorescence imaging conducted on mouse blood samples immediately after injection, and then 1, 3, 5, and 7 days after injection show a significant decrease over time of the fluorescence signal in the control group compared to the other experimental groups where fluorescence levels are sustained. These results indicate the increased in vivo viability of allogeneic Tregs expressing the membrane-bound polypeptides described in the present disclosure, compared to control allogeneic Tregs.

Example 5: Expression of CD2-binding membrane-bound polypeptides in vitro

293T cells were transfected with pcDNA vectors encoding CD2-binding membranebound polypeptides shown in FIG. 14 and Table 2 using the TransIT-2020 reagent. Control cells were treated with the TransIT-2020 reagent alone. Two to three days post-transfection, expression of the CD2-binding membrane-bound polypeptides was detected by flow cytometry using antibodies targeting the CD22, CD45RO, or CD45ABC domains. The frequency of CD2-binding membrane-bound polypeptides expression on transfected cells was quantified from four replicates as seen in FIG. 15 A. A representative flow plot for the 1 (+)22 CD2-binding membrane-bound polypeptide is shown in FIG. 15B. The data demonstrate that the CD2-binding membrane-bound polypeptides were successfully expressed on human cells.

Example 6: Effects of CD2-binding membrane-bound polypeptides on antigen specific T cell response

Purified pan T cells were transduced with either the HLA-A2 restricted A6 or 1G4 T cell receptors (TCRs). TCR-engineered effector T cells were then co-cultured with 293T cells (naturally HLA-A2+) transfected with the indicated CD2-binding membrane-bound polypeptides (see FIG. 14 and Table 2) and the cognate epitope peptide. Response was measured by upregulation of surface activation markers by flow cytometry after 22 hours. CD137 expression level on CD8+ TCR-engineered T cells were quantified and normalized to that of the response against control 293T cell targets as seen FIG. 16A. FIG. 16B shows representative flow plots of CD8+ effector T cell responses. These results indicate that expression of all CD2-binding membrane-bound polypeptides reduced the T cell response, as evidenced by the reduction of surface activation markers.

Example 7: Effects of CD2-binding membrane-bound polypeptides on allogenic T cell response

Purified pan T cells from healthy donors were labeled with carboxyfluorescein succinimidyl ester (CFSE) and co-cultured with irradiated control 293T cells or 293T cells transfected with the indicated CD2-binding membrane-bound polypeptides (see FIG. 14 and Table 2). Control cells were treated with transfection reagent alone. T cells were stimulated with anti-CD3/CD28 Dynabeads as a positive control. CFSE dilution among CD8+ cells was measured by flow cytometry after 5-6 days of co-culture. The frequency of CD8+ CFSE-low cells were quantified across three replicates as seen in FIG. 17A. FIG. 17B shows representative CFSE plots with CSFE-low (lower band) and CSFE-high (upper band) gates shown. These results demonstrate that expression of CD2-binding membrane-bound polypeptides reduced alloreactive T cell responses.

Table 1: Exemplary domains of the membrane-bound polypeptides as provided in the

Table 2. Exemplary Designs of Constructs (see Examples 5-7; FIGs. 14-17)

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

Claims

CLAIMS What is claimed is:

1. A membrane-bound polypeptide comprising:

(a) an extracellular CD2-binding domain comprising a modified CD2-binding domain of CD58, wherein the modified CD2-binding domain of CD58 comprises one or more amino acid substitutions relative to SEQ ID NO: 2, or a CD2-binding single-chain variable fragment (scFv) ;

2. The membrane-bound polypeptide of claim 1, further comprising (d) an intracellular domain.

3. The membrane-bound polypeptide of claim 2, wherein (a)-(d) are connected from N- terminus to C-terminus in the following order: intracellular domain, transmembrane domain, extracellular elongation domain, and extracellular CD2-binding domain.

4. The membrane -bound polypeptide of any one of the preceding claims, wherein the extracellular CD2-binding domain is connected to the extracellular elongation domain by a linker.

5. The membrane-bound polypeptide of any one of the preceding claims, wherein the one or more amino acid substitutions relative to SEQ ID NO: 2 are substitutions at E25, K29, K32, D33, E37 and/or R44 of SEQ ID NO: 2.

6. The membrane-bound polypeptide of claim 5, wherein the one or more amino acid substitutions relative to SEQ ID NO: 2 are E25A, K29A, K32A, D33A, E37A and/or R44A relative to SEQ ID NO: 2.

7. The membrane -bound polypeptide of any one of the preceding claims, wherein the extracellular CD2-binding domain comprises any one of SEQ ID NOs: 3-8.

8. The membrane-bound polypeptide of any one of the preceding claims, further comprising a second extracellular CD2-binding domain comprising a modified CD2-binding domain of CD58, wherein the modified CD2-binding domain of CD58 comprises one or more amino acid substitutions relative to SEQ ID NO: 2.

9. The membrane-bound polypeptide of any one of the preceding claims, wherein the at least one rigid protein module comprises an Ig-like domain of CD22.

10. The membrane -bound polypeptide of any one of the preceding claims, wherein the extracellular elongation domain comprises a first rigid protein module and a second rigid protein module, wherein the first rigid protein module is N-term relative to the second rigid protein module, and wherein the first rigid protein module comprises a sequence that is at least 85% identical to the C6 Ig-like domain of CD22 as set forth in SEQ ID NO: 29, and the second rigid protein module comprises a sequence that is at least 85% identical to the C5 Ig-like domain of CD22 as set forth in SEQ ID NO: 28.

11. The membrane -bound polypeptide of claim 10, wherein the extracellular elongation domain further comprises a third rigid protein module that is C-term relative to the second rigid protein module, wherein the third rigid protein module comprises a sequence that is at least 85% identical to the C4 Ig-like domain of CD22 as set forth in SEQ ID NO: 27.

12. The membrane -bound polypeptide of claim 11, wherein the extracellular elongation domain further comprises a fourth rigid protein module that is C-term relative to the third rigid protein module, wherein the fourth rigid protein module comprises a sequence that is at least 85% identical to the C3 Ig-like domain of CD22 as set forth in SEQ ID NO: 26.

13. The membrane -bound polypeptide of claim 12, wherein the extracellular elongation domain further comprises a fifth rigid protein module that is C-term relative to the fourth rigid protein module, wherein the fifth rigid protein module comprises a sequence that is at least 85% identical to the C2 Ig-like domain of CD22 as set forth in SEQ ID NO: 25.

14. The membrane -bound polypeptide of any one of the preceding claims, wherein the extracellular CD2-binding domain is linked to the extracellular elongation domain via a linker comprising the amino acid sequence of SEQ ID NO: 1.

15. A membrane-bound polypeptide comprising:

(a) an extracellular CD2-binding domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-8;

(b) an extracellular elongation domain comprising at least one rigid protein module comprising an Ig-like domain of CD22; and

16. The membrane-bound polypeptide of claim 15, further comprising (d) an intracellular domain.

17. The membrane-bound polypeptide of claim 16, wherein (a)-(d) are connected from N- terminus to C-terminus in the following order: intracellular domain, transmembrane domain, extracellular elongation domain, and extracellular CD2-binding domain.

18. The membrane-bound polypeptide of claim 15 or 16, wherein the extracellular CD2- binding domain comprises any one of SEQ ID NOs: 3-8.

19. The membrane-bound polypeptide of any one of claims 15-18, further comprising a second extracellular CD2-binding domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-8.

20. The membrane-bound polypeptide of any one of claims 15-19, wherein the extracellular elongation domain comprises a first rigid protein module and a second rigid protein module, wherein the first rigid protein module is N-term relative to the second rigid protein module, and wherein the first rigid protein module comprises a sequence that is at least 85% identical to the C6 Ig-like domain of CD22 as set forth in SEQ ID NO: 29, and the second rigid protein module comprises a sequence that is at least 85% identical to the C5 Ig-like domain of CD22 as set forth in SEQ ID NO: 28.

21. The membrane -bound polypeptide of claim 20, wherein the extracellular elongation domain further comprises a third rigid protein module that is C-term relative to the second rigid protein module, wherein the third rigid protein module comprises a sequence that is at least 85% identical to the C4 Ig-like domain of CD22 as set forth in SEQ ID NO: 27.

22. The membrane -bound polypeptide of claim 21, wherein the extracellular elongation domain further comprises a fourth rigid protein module that is C-term relative to the third rigid protein module, wherein the fourth rigid protein module comprises a sequence that is at least 85% identical to the C3 Ig-like domain of CD22 as set forth in SEQ ID NO: 26.

23. The membrane -bound polypeptide of claim 22, wherein the extracellular elongation domain further comprises a fifth rigid protein module that is C-term relative to the fourth rigid protein module, wherein the fifth rigid protein module comprises a sequence that is at least 85% identical to the C2 Ig-like domain of CD22 as set forth in SEQ ID NO: 25.

24. A membrane-bound polypeptide comprising an extracellular CD2-binding domain comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 2-8; and a CD22 stalk comprising an extracellular elongation domain, a transmembrane domain, and optionally an intracellular domain sequence.

25. The membrane-bound polypeptide of claim 24, wherein the CD22 stalk comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 9-12.

26. The membrane-bound polypeptide of claim 24 or 25, wherein the extracellular CD2- binding domain is linked to the CD22 stalk directly, via a linker comprising the amino acid sequence of SEQ ID NO: 1, or via a CD8 linker.

27. The membrane-bound polypeptide of any one of the preceding claims, comprising a sequence that is at least 85% identical to any one of SEQ ID NOs: 13-22.

28. A nucleic acid comprising a sequence encoding the membrane-bound polypeptide of any one of the preceding claims.

29. A vector comprising the nucleic acid of claim 28.

30. The vector of claim 29, further comprising a promoter operably linked to the nucleic acid sequence encoding the membrane-bound polypeptide.

31. A method for producing a therapeutic cell capable of evading a host immune system, the method comprising inserting into the cell the nucleic acid of claim 28, contacting the cell with the vector of claim 29 or 30, or expressing in the cell the membrane-bound polypeptide of any one of claims 1-27.

32. A cell expressing the membrane -bound polypeptide of any one of claims 1-27, or comprising the nucleic acid of claim 28 or the vector of claim 29 or 30.

33. The cell of claim 32, wherein the cell is a T cell, optionally a Treg.

34. The cell of claim 33, wherein the T cell expresses a chimeric antigen receptor (CAR) or a TCR.

35. A method comprising administering to a subject the cell of any one of claims 32-34.

36. A membrane-bound polypeptide comprising:

(a) an extracellular CD28-binding domain; (b) an extracellular elongation domain comprising at least one rigid protein module; and

(c) a transmembrane domain, wherein (a)-(c) are connected from N-terminus to C-terminus in the following order: transmembrane domain, extracellular elongation domain, and extracellular CD28-binding domain.

37. The membrane-bound polypeptide of claim 36, further comprising (d) an intracellular domain.

38. The membrane-bound polypeptide of claim 37, wherein (a)-(d) are connected from N- terminus to C-terminus in the following order: intracellular domain, transmembrane domain, extracellular elongation domain, and extracellular CD28-binding domain.

39. The membrane-bound polypeptide of any one of claims 36-38, wherein the extracellular CD28-binding domain is connected to the extracellular elongation domain by a linker.

40. The membrane-bound polypeptide of any one of claims 36-39, wherein the extracellular CD28-binding domain comprises a CD28-binding domain of a B7 costimulatory ligand.

41. The membrane-bound polypeptide of claim 40, wherein the B7 costimulatory ligand is CD80.

42. The membrane-bound polypeptide of claim 41 , wherein the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD80 CD28-binding domain as set forth in SEQ ID NO: 34.

43. The membrane-bound polypeptide of claim 40, wherein the B7 costimulatory ligand is CD86.

44. The membrane-bound polypeptide of claim 43, wherein the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD86 CD28-binding domain as set forth in SEQ ID NO: 35.

45. The membrane-bound polypeptide of any one of claims 36-44, wherein the at least one rigid protein module comprises an Ig-like domain of CD22.

46. The membrane-bound polypeptide of any one of claims 36-45, wherein the extracellular elongation domain comprises a first rigid protein module and a second rigid protein module, wherein the first rigid protein module is N-term relative to the second rigid protein module, and wherein the first rigid protein module comprises a sequence that is at least 85% identical to the C6 Ig-like domain of CD22 as set forth in SEQ ID NO: 29, and the second rigid protein module comprises a sequence that is at least 85% identical to the C5 Ig-like domain of CD22 as set forth in SEQ ID NO: 28.

47. The membrane -bound polypeptide of claim 46, wherein the extracellular elongation domain further comprises a third rigid protein module that is C-term relative to the second rigid protein module, wherein the third rigid protein module comprises a sequence that is at least 85% identical to the C4 Ig-like domain of CD22 as set forth in SEQ ID NO: 27.

48. The membrane -bound polypeptide of claim 47, wherein the extracellular elongation domain further comprises a fourth rigid protein module that is C-term relative to the third rigid protein module, wherein the fourth rigid protein module comprises a sequence that is at least 85% identical to the C3 Ig-like domain of CD22 as set forth in SEQ ID NO: 26.

49. The membrane -bound polypeptide of claim 48, wherein the extracellular elongation domain further comprises a fifth rigid protein module that is C-term relative to the fourth rigid protein module, wherein the fifth rigid protein module comprises a sequence that is at least 85% identical to the C2 Ig-like domain of CD22 as set forth in SEQ ID NO: 25.

50. The membrane-bound polypeptide of any one of claims 36-49, wherein the extracellular CD28-binding domain is linked to the extracellular elongation domain via a linker comprising the amino acid sequence of SEQ ID NO: 1.

51. A membrane-bound polypeptide comprising: an extracellular CD28-binding domain and a CD22 stalk comprising an extracellular elongation domain, a transmembrane domain, and optionally an intracellular domain sequence.

52. The membrane-bound polypeptide of claim 51, wherein the CD22 stalk comprises a sequence that is at least 85% identical to SEQ ID NO: 9.

53. The membrane-bound polypeptide of claim 51 or 52, wherein the extracellular CD28- binding domain comprises a CD28-binding domain of a B7 costimulatory ligand.

54. The membrane-bound polypeptide of claim 53, wherein the B7 costimulatory ligand is CD80.

55. The membrane-bound polypeptide of claim 54, wherein the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD80 CD28-binding domain as set forth in SEQ ID NO: 34.

56. The membrane-bound polypeptide of claim 53, wherein the B7 costimulatory ligand is CD86.

57. The membrane-bound polypeptide of claim 56, wherein the extracellular CD28-binding domain comprises a sequence that is at least 85% identical to the CD86 CD28-binding domain as set forth in SEQ ID NO: 35.

58. The membrane -bound polypeptide of any one of claims 36-57, having a sequence identical to SEQ ID NO: 40 or 41.

59. A nucleic acid comprising a sequence encoding the membrane-bound polypeptide of any one of claims 36-58.

60. A vector comprising the nucleic acid of claim 59.

61. The vector of claim 60, further comprising a promoter operably linked to the nucleic acid sequence encoding the membrane-bound polypeptide.

62. A method for producing a therapeutic cell capable of evading a host immune system, the method comprising inserting into the cell the nucleic acid of claim 59, contacting the cell with the vector of claim 60 or 61, or expressing in the cell the membrane-bound polypeptide of any one of claims 36-58.

63. A cell expressing the membrane-bound polypeptide of any one of claims 36-58, or comprising the nucleic acid of claim 59 or the vector of claim 60 or 61.

64. The cell of claim 63, wherein the cell is a T cell, optionally a Treg.

65. The cell of claim 64, wherein the T cell expresses a chimeric antigen receptor (CAR) or a TCR.

66. A method comprising administering to a subject the cell of any one of claims 63-65.