CN116804060A - anti-BCMA/anti-P329G bispecific antibody, chimeric antigen receptor binding to anti-P329G and application thereof - Google Patents

Abstract

The present invention relates to anti-BCMA/anti-P329G bispecific antibodies that specifically bind to B cell maturation antigen protein (BCMA) and specifically bind to P329G mutation, nucleic acids encoding the anti-BCMA/anti-P329G bispecific antibodies, and comprising the nucleic acids Host cells and methods for preparing the anti-BCMA/anti-P329G bispecific antibodies. The invention also relates to chimeric antigen receptor (CAR) polypeptides that bind anti-P329G, nucleic acids encoding the CAR polypeptides, cells expressing the CAR polypeptides, and methods of preparing the cells. The present invention also relates to a pharmaceutical combination of the anti-BCMA/anti-P329G bispecific antibody and immune effector cells (eg, T cells, NK cells) expressing the CAR polypeptide, and relates to the pharmaceutical combination for the treatment of patients with BCMA. Expression-related diseases, such as cancers that express or overexpress BCMA.

Description

Anti-BCMA/anti-P329G bispecific antibodies, chimeric antigen receptors binding to anti-P329G and their applications

Technical Field

The present invention generally relates to the fields of antibody engineering and cellular immunology. Specifically, the present invention relates to an anti-BCMA/anti-P329G bispecific antibody that specifically binds to B-cell maturation antigen protein (BCMA) and specifically binds to the P329G mutation, to a chimeric antigen receptor that binds to anti-P329G, to a combination of the anti-BCMA/anti-P329G bispecific antibody and immune effector cells (e.g., T cells, NK cells) engineered to express a chimeric antigen receptor that binds to anti-P329G, and to the use of the combination for treating diseases associated with the expression of BCMA, such as cancers that express or overexpress BCMA.

Background Art

B cell maturation antigen (BCMA, i.e. CD269, TNFRSF17) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). BCMA is a type III transmembrane protein with a cysteine-rich domain (CRD) characteristic of TNFR family members in the extracellular domain (ECD), which forms a ligand binding motif. BCMA's ligands include B cell activating factor (BAFF) and B cell proliferation inducing ligand (APRIL), of which B cell proliferation inducing ligand (APRIL) binds to BCMA with higher affinity and promotes tumor cell proliferation.

BCMA is mainly expressed on the surface of mature B cells, i.e. plasma cells, and is not expressed in normal hematopoietic stem cells and non-hematopoietic tissues. BCMA signaling is essential for the survival of long-term bone marrow plasma cells, but is not necessary for overall B cell homeostasis. BCMA on the membrane surface can be cleaved and shed by γ-secretase, and the resulting soluble BCMA (sBCMA) may reduce the signal transduction of BCMA on the membrane surface by blocking BAFF/APRIL ligand binding. In preclinical models and human tumors, BCMA was found to be overexpressed in multiple myeloma (MM) cells, which upregulated classical and non-classical NF-κB signals, promoted MM cell growth, survival, adhesion, induced osteoclast activation, angiogenesis, metastasis and immunosuppression, etc. BCMA expression has become an important marker for the diagnosis of MM. In addition, the level of sBCMA in the serum of MM patients is increased, which is positively correlated with the number of MM cells in the bone marrow, and its concentration changes are closely related to MM prognosis and treatment response.

Given that BCMA is only expressed in plasma cells and not in natural and memory B cells, BCMA has become a popular target for the treatment of MM. A variety of targeted drugs have been developed, including chimeric antigen receptor T cell (CAR-T) immunotherapy. Among them, Bluebird's Abecma (idecabtagene vicleucel, ide-cel) was approved by the FDA in March 2021 for the treatment of 4-line and above relapsed and refractory MM (RRMM). Ciltacabtagene autoleucel (Cilta-cel) jointly developed by Johnson & Johnson and Nanjing Legend has also obtained the FDA Biologics License Approval (BLA).

Abecma is the first CAR-T cell therapy targeting BCMA. It recognizes and binds to the BCMA protein on multiple myeloma cancer cells, causing the death of cancer cells expressing the BCMA protein. That is, it directly targets the surface antigen BCMA protein of tumor cells through the chimeric antigen receptor (CAR) on the CAR-T cell, thereby achieving the purpose of identifying and killing tumor cells. The disadvantage of the traditional chimeric antigen receptor T cell is that there is a lack of control over the activity of CAR-T cells after infusion. After activation in vivo, these CAR-T cells will continue to recognize and kill cells expressing their targeted antigens. In some cases (such as large tumors in tumor patients), a large number of rapidly activated CAR-T cells will release massive inflammatory cytokines, causing patients to have cytokine release syndrome (CRS), neurotoxicity (NT) and other serious side effects. A meta-study of 84 clinical trials with 2,592 patients showed that the incidence of CRS and NT above grade 3 after receiving traditional CAR-T cell therapy was 29% and 28%, respectively. Although a large number of clinical trials have found that most patients who experience toxic reactions can recover quickly after receiving corresponding treatments and do not significantly affect the anti-tumor efficacy, the treatment of toxic and side effects takes up a lot of clinical resources and increases the physical, mental and economic burden on patients.

Since most CAR-T cells (e.g., Abecma) target antigens that are tumor-associated antigens (TAAs) rather than tumor-specific antigens (TSAs), these TAAs are often expressed at low levels in many normal tissues, especially important tissues and organs, in addition to being expressed on tumor cells. The recognition and killing of the normal tissues by CAR-T cells leads to "on-target/off tumor" toxicity, which the body may be able to tolerate in the short term, but if it cannot be recovered or alleviated for a long time, it may cause serious side effects. For example, CAR-T cells targeting CD19 are used clinically to treat CD19-positive blood tumors. In addition to clearing tumor cells, CD19 CAR-T cells also kill normal B cells. Although the continued presence of CAR-T cells in the body is closely related to the patient's long-term recurrence-free survival, it also leads to long-term B cell dysplasia and lack of humoral immunity in the body, which is prone to infection. Clinical trials have shown that within 30 days after receiving CD19 CAR-T cell therapy, 27-36% of lymphoma patients developed bacterial infections, 9.2-28% of patients developed viral infections after one month, and it took a median of 6.7 months for B cells to recover. 31-64% of patients required immunoglobulin replacement therapy. In addition, continuous activation of CAR-T cells in the body can easily lead to functional exhaustion, impairing their anti-tumor effects and reducing their persistence in the body, thereby reducing long-term treatment effects.

In order to reduce the "on-target/off tumor" toxicity of CAR-T cells, the following strategies are generally used in the prior art. One strategy is to design the antigen binding domain contained in the CAR to target the target antigen that is highly expressed on the tumor surface but not expressed or lowly expressed in normal tissues during CAR design. Another strategy is to strictly control the dose of T cells administered, because too many CAR-T cells will increase exponentially after being stimulated by antigens, which is more likely to cause on-target/off-tumor effects. Another strategy is to introduce an inducible suicide gene, such as the inducible Caspase-9 (iCasp9) suicide gene, when constructing CAR. When patients are observed to experience on-target/off-tumor toxicity, AP1903 (a dimerization chemical inducer that can activate iCasp9) is administered to induce apoptosis of CAR-T cells and reduce toxicity (Zhang Huihui et al., Preclinical study of suicide genes as a "safety switch" to control CAR-T cell toxicity, Chinese Journal of Oncology Biotherapy, 2021, 28(3): 225-231). There are also reports of using the herpes simplex virus thymidine kinase (HSV-TK) gene as a suicide gene to induce apoptosis of CAR-T cells by administering ganciclovir. In addition, there are also reports using electroporation transfection methods to make T cells transiently express CAR, exerting therapeutic effects through transient killing function (Kenderian SS et al., CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia [J] Leukemia. 2015; 29(8): 1637-1647).

Regarding the schemes reported in the prior art that use suicide genes as a "switch" to control the "on-target/off tumor" toxicity of CAR-T, when iCasp9 is introduced into CAR-T cells, AP1903 is an experimental drug and the experimenter must sign an agreement before it can be obtained through the supplier; when the HSV-TK gene is introduced into CAR-T cells, although ganciclovir has been used to control graft-versus-host disease in stem cell transplantation and has achieved certain clinical results, the drug itself is immunogenic and leads to the elimination of cells other than target cells (Traversari C et al., The potential immunogenicity of the TK suicide gene does not prevent full clinical benefit associated with the use of TK-transduced donor lymphocytes in HSCT for hematologic malignancies [J] Blood. 2007; 109(11): 4708-4715). In addition, the mechanism of TK action is to interfere with DNA synthesis during cell mitosis and only acts on dividing cells, requiring several days or even weeks to achieve the desired effect.

Therefore, there is still an urgent need in this field to develop other "molecular switches" to regulate the activity of CAR-T cells.

SUMMARY OF THE INVENTION

Traditional CAR-T cells targeting BCMA still face toxicity and relapse problems in the treatment of MM. A meta-study showed that the incidence of CRS and NT after receiving traditional CAR-T cell therapy targeting BCMA was 74% (95% CI, 56-91%) and 34% (95% CI, 24-43%), of which the incidence of grade 3 and above events was 25% (95% CI, 7-43%) and 12% (95% CI, 4-20%), respectively. In addition, the humoral immunity of the treated patients was impaired due to the loss of plasma cells in the body, and the incidence of infection increased. Among the 128 patients with relapsed and refractory multiple myeloma (RRMM) treated with ide-cel, 69% had infection, and the incidence of grade 3 and above infection was 22%. Most patients needed to receive anti-infection, white blood cell growth factor and immunoglobulin replacement therapy. Similarly, among the 97 RRMM patients treated with Cilta-cel, 56% and 20% had infection and grade 3 and above infection. These clinical results suggest the need to develop a regulatable BCMA-targeted CAR-T cell therapy.

The inventors have designed and constructed the P329G CAR-T cells of the present invention (i.e., T cells expressing CARs containing P329G mutations in the extracellular region) that target BCMA by binding to anti-BCMA/anti-P329G bispecific antibody molecules that specifically bind to BCMA and P329G mutations, and verified their specific and controllable anti-tumor effects. Different from traditional BCMA-targeted CAR-T cells that directly recognize and kill MM cells, the drug combination of the present invention includes two components: an anti-BCMA/anti-P329G bispecific antibody that specifically binds to BCMA and P329G mutations, and a CAR-T cell containing P329G mutations. The drug combination utilizes the anti-BCMA portion of the anti-BCMA/anti-P329G bispecific antibody to recognize tumor cells expressing BCMA. At the same time, the anti-P329G portion of the anti-BCMA/anti-P329G bispecific antibody recruits the CAR-T cells of the present invention to tumor cells expressing BCMA by binding to the P329G mutation contained in the extracellular region of the CAR-T cells of the present invention, thereby generating tumor recognition and killing effects (see Figure 1). In the treatment method using the drug combination of the present invention, the anti-BCMA/anti-P329G bispecific antibody molecule that specifically targets BCMA and specifically binds to the P329G mutation serves as a bridge connecting the P329G CAR-T cells of the present invention and tumor cells expressing BCMA, plays a "molecular switch" role, and regulates the activity of the P329G CAR-T cells of the present invention.

Therefore, in a first aspect, the present invention provides an anti-BCMA/anti-P329G bispecific antibody capable of specifically binding to BCMA and specifically binding to the P329G mutation as a "molecular switch" for immune effector cells (e.g., T cells, NK cells) expressing a CAR polypeptide of the present invention, which comprises two heavy chains and two light chains, and

i) having a F(ab) ₂ that specifically binds to BCMA, and an anti-P329G heavy chain variable region that specifically binds to the P329G mutation located at the C-terminus of the CH3 domain of one heavy chain, and an anti-P329G light chain variable region that specifically binds to the P329G mutation located at the C-terminus of the CH3 domain of the other heavy chain; or

ii) having two half antibodies consisting of one heavy chain and one light chain, wherein one half antibody has an N-terminal Fab that specifically binds to BCMA and a heavy chain variable region that specifically binds to the anti-P329G mutation located between the C-terminus of the heavy chain Fab and the N-terminus of the CH2 domain, and the other half antibody has an N-terminal Fab that specifically binds to BCMA and a light chain variable region that specifically binds to the anti-P329G mutation located between the C-terminus of the heavy chain Fab and the N-terminus of the CH2 domain; or

iii) an anti-P329G scFv having F(ab) ₂ that specifically binds to BCMA and specifically binds to the P329G mutation, wherein the C-terminus of the CH3 domain of one heavy chain is connected to the N-terminus of the anti-P329G scFv.

In some embodiments, the present invention obtains an anti-BCMA/anti-P329G bispecific antibody having the above three composition forms, wherein

a) The Fab or F(ab) ₂ that specifically binds to BCMA comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR H1 shown in the amino acid sequence SSSYYWT (SEQ ID NO: 25) according to Kabat numbering, or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR H2 shown in the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR H3 shown in the amino acid sequence DRGDQILDV (SEQ ID NO: 27), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises CDR L1 shown in the amino acid sequence RASQSISRYLN (SEQ ID NO: 28) according to Kabat numbering, or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR L2, or a variant of said CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR L3 shown in the amino acid sequence QQKYFDIT (SEQ ID NO: 30), or a variant of said CDR L3 with no more than 2 amino acid changes or no more than 1 amino acid change;

wherein the amino acid change is an addition, deletion or substitution of an amino acid; and

b) the anti-P329G or anti-P329G scFv that specifically binds to the P329G mutation comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementary determining region CDR H1 as shown in the amino acid sequence RYWMN (SEQ ID NO: 31) according to Kabat numbering, or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; a CDR H2 as shown in the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 32), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and a CDR H3 as shown in the amino acid sequence PYDYGAWFAS (SEQ ID NO: 33), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises a light chain complementary determining region (CDR L) 1 as shown in the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 34) according to Kabat numbering, or a variant of the CDR CDR L1 having no more than 2 amino acid changes or no more than 1 amino acid change; CDR L2 shown in the amino acid sequence GTNCRAP (SEQ ID NO: 35), or a variant of said CDR L2 having no more than 2 amino acid changes or no more than 1 amino acid change; and CDR L3 shown in the amino acid sequence ALWYSNHWV (SEQ ID NO: 36), or a variant of said CDR L3 having no more than 2 amino acid changes or no more than 1 amino acid change;

The amino acid change is an addition, deletion or substitution of an amino acid.

In some embodiments, the present invention provides an anti-BCMA/anti-P329G bispecific antibody having the above three composition forms, wherein:

a) the Fab or F(ab) ₂ that specifically binds to BCMA comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR H1 shown by the amino acid sequence SSSYYWT (SEQ ID NO: 25) according to Kabat numbering; CDR H2 shown by the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26); and CDR H3 shown by the amino acid sequence DRGDQILDV (SEQ ID NO: 27); the light chain variable region comprises CDR L1 shown by the amino acid sequence RASQSISRYLN (SEQ ID NO: 28) according to Kabat numbering; CDR L2 shown by the amino acid sequence AASSLQS (SEQ ID NO: 29); and CDR L3 shown by the amino acid sequence QQKYFDIT (SEQ ID NO: 30); and

b) The anti-P329G or anti-P329G scFv that specifically binds to the P329G mutation comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR H1 shown by the amino acid sequence RYWMN (SEQ ID NO: 31) according to Kabat numbering; CDR H2 shown by the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 32); and CDR H3 shown by the amino acid sequence PYDYGAWFAS (SEQ ID NO: 33); the light chain variable region comprises CDR L1 shown by the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 34) according to Kabat numbering; CDR L2 shown by the amino acid sequence GTNCRAP (SEQ ID NO: 35); and CDR L3 shown by the amino acid sequence ALWYSNHWV (SEQ ID NO: 36).

In some embodiments, the present invention obtains an anti-BCMA/anti-P329G bispecific antibody having the above three composition forms, wherein:

a) the Fab or F(ab) ₂ that specifically binds to BCMA comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereof, and the light chain variable region comprising the sequence of SEQ ID NO: 24, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereof; and

b) the anti-P329G or anti-P329G scFv that specifically binds to the P329G mutation comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the sequence of SEQ ID NO: 21, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the light chain variable region comprising the sequence of SEQ ID NO: 22, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the present invention obtains an anti-BCMA/anti-P329G bispecific antibody having the above three composition forms, wherein:

a) the Fab or F(ab) ₂ that specifically binds to BCMA comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the sequence of SEQ ID NO: 23, and the light chain variable region comprises the sequence of SEQ ID NO: 24; and

b) the anti-P329G or anti-P329G scFv that specifically binds to the P329G mutation comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the sequence of SEQ ID NO: 21, and the light chain variable region comprises the sequence of SEQ ID NO: 22.

In some embodiments, the anti-BCMA/anti-P329G bispecific antibody of the present invention is of IgG1 or IgG4 class; preferably, it is of IgG1 class.

In some embodiments, the two heavy chains of the anti-BCMA/anti-P329G bispecific antibody of the present invention comprise a protrusion or a cavity in each Fc region, respectively, and the protrusion or cavity in the Fc region of one heavy chain can be placed in the cavity or protrusion in the Fc region of the other heavy chain, respectively, so that the heavy chains of the anti-BCMA/anti-P329G bispecific antibody form a "knotted" stable association with each other.

In some embodiments, the anti-BCMA/anti-P329G bispecific antibody of the present invention comprises:

i) two light chains as set forth in SEQ ID NO: 2 and two heavy chains as set forth in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, or sequences at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to any of said sequences; or

ii) two light chains as set forth in SEQ ID NO: 2 and two heavy chains as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or sequences at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to any of said sequences; or

iii) two light chains as set forth in SEQ ID NO:2 and two heavy chains as set forth in SEQ ID NO:6 and SEQ ID NO:7, respectively, or sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to any of said sequences.

In a second aspect, the present invention provides a nucleic acid encoding the anti-BCMA/anti-P329G bispecific antibody of the first aspect of the present invention, a vector comprising a nucleic acid encoding the anti-BCMA/anti-P329G bispecific antibody, a cell comprising the nucleic acid molecule or the vector, and a method for preparing the anti-BCMA/anti-P329G bispecific antibody.

In some embodiments, the method for preparing the anti-BCMA/anti-P329G bispecific antibody comprises culturing a host cell into which an expression vector encoding the anti-BCMA/anti-P329G bispecific antibody of the first aspect of the present invention is introduced under conditions suitable for expressing the nucleic acid encoding the anti-BCMA/anti-P329G bispecific antibody of the first aspect of the present invention, isolating the anti-BCMA/anti-P329G bispecific antibody, and optionally the method further comprises recovering the anti-BCMA/anti-P329G bispecific antibody from the host cell. Preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies, and most preferably, the host cell is a HEK293 cell or a CHO cell.

In some embodiments, the method for preparing the anti-BCMA/anti-P329G bispecific antibody comprises culturing a host cell introduced with an expression vector encoding a nucleic acid encoding one of the two half antibodies of the anti-BCMA/anti-P329G bispecific antibody of the first aspect of the invention under conditions suitable for expressing nucleic acids encoding the two half antibodies in the anti-BCMA/anti-P329G bispecific antibody of the first aspect of the invention, isolating one half antibody of the anti-BCMA/anti-P329G bispecific antibody, and culturing a host cell introduced with an expression vector encoding a nucleic acid encoding the other half antibody of the two half antibodies of the anti-BCMA/anti-P329G bispecific antibody of the first aspect of the invention, isolating the other half antibody of the anti-BCMA/anti-P329G bispecific antibody, and optionally the method further comprises recovering the two half antibodies from the host cell; the combination of the two half antibodies constitutes the anti-BCMA/anti-P329G bispecific antibody.

In a third aspect, the present invention provides a molecular switch-regulated chimeric antigen receptor (CAR) polypeptide, which is a chimeric antigen receptor (CAR) polypeptide that binds to anti-P329G, and the chimeric antigen receptor polypeptide comprises:

(i) an extracellular domain, which is a mutant Fc domain or a fragment thereof or a combination of fragments thereof, wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G);

For example, the mutant Fc domain is a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody, preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 antibody;

(ii) a CD8 transmembrane domain or a variant thereof having 1-10 amino acid modifications;

(iii) a 4-1BB co-stimulatory domain or a variant thereof having 1 to 10 amino acid modifications; and

(iv) CD3ζ activation domain or a variant thereof having 1-10 amino acid modifications.

In some embodiments, the present invention provides a molecular switch-regulated chimeric antigen receptor (CAR) polypeptide, which is a chimeric antigen receptor (CAR) polypeptide that binds to anti-P329G, wherein the extracellular domain of the chimeric antigen receptor polypeptide is a mutant Fc domain fragment or a combination of its fragments, for example, a single CH2 domain comprising a P329G mutation; a single CH2 domain comprising a P329G mutation and a truncated N-terminal portion; a CH2CH2 domain composed of two CH2 domains comprising a P329G mutation; a CH2CH3 domain composed of a CH2 domain containing a P329G mutation and a normal CH3 domain connected; or a single CH2 domain comprising P329G, N297G mutations; but not limited thereto.

In some embodiments, the chimeric antigen receptor of the present invention further comprises a G4S linker (SEQ ID NO: 17) between (i) and (ii); and/or a signal peptide sequence located at the N-terminus of (i), for example, the signal peptide sequence shown in SEQ ID NO: 16.

In some embodiments, the chimeric antigen receptor polypeptide of the present invention comprises:

(i) a P329G mutant Fc domain as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, or a fragment thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or a combination of fragments thereof;

(ii) a CD8 transmembrane domain as shown in SEQ ID NO: 18 or a variant thereof having 1-5 amino acid modifications;

(iii) a 4-1BB co-stimulatory domain as shown in SEQ ID NO: 19 or a variant thereof having 1 to 5 amino acid modifications; and

(iv) the CD3ζ activation domain as shown in SEQ ID NO: 20 or a variant thereof having 1-5 amino acid modifications.

In a fourth aspect, the present invention provides a nucleic acid encoding a molecular switch-regulated CAR polypeptide as described herein, a vector comprising a nucleic acid encoding a CAR polypeptide as described herein, and a cell comprising a CAR nucleic acid molecule or vector as described herein, or a cell expressing a CAR polypeptide as described herein, preferably, the cell is an autologous T cell or an allogeneic T cell.

In some embodiments, the vector comprising a nucleic acid encoding a CAR polypeptide described herein is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

In one embodiment, the present invention uses human peripheral blood mononuclear cells (PBMC) to prepare primary P329G CAR-T cells. CAR-T cells transduced with the CAR molecules of the present invention have in vitro effector functions and have the activity of killing target cells in vitro.

In a fifth aspect, the present invention provides a method for producing cells, such as immune effector cells, the method comprising introducing (e.g., transducing) a nucleic acid molecule (e.g., an RNA molecule, such as an mRNA molecule) encoding a CAR polypeptide described herein, or a vector comprising a nucleic acid molecule encoding a CAR polypeptide described herein into an immune effector cell.

In some embodiments, the immune effector cells are T cells, NK cells, for example, the T cells are autologous T cells or allogeneic T cells, for example, the immune effector cells are prepared by isolating T cells, NK cells from human PBMCs.

In a sixth aspect, the present invention provides a pharmaceutical combination comprising

(i) a nucleic acid molecule encoding a CAR polypeptide according to the fourth aspect of the present invention, a vector comprising the nucleic acid molecule, a cell, and any combination thereof; and

(ii) the anti-BCMA/anti-P329G bispecific antibody according to the first aspect of the present invention; and

Optionally, pharmaceutically acceptable excipients are included.

In some embodiments, the immune effector cells in the drug combination of the present invention are autologous T cells, NK cells or allogeneic T cells, NK cells prepared from the CAR polypeptide of the present invention. For example, the immune effector cells are T cells, NK cells expressing the CAR polypeptide of the present invention prepared from T cells, NK cells isolated from human PBMC.

In some embodiments, the present invention utilizes primary CAR-T cells prepared from human PBMCs from a donor source, and utilizes anti-BCMA/anti-P329G bispecific antibodies to evaluate the effector function of the CAR-T cells of the present invention against BCMA-expressing tumor cells through anti-BCMA/anti-P329G bispecific antibodies in an in vitro co-culture system. Thus, the anti-BCMA/anti-P329G bispecific antibodies can act as a "molecular switch" to regulate the recognition and killing activity of BCMA-positive tumor cells by CAR-T cells bound to anti-P329G. This in vitro effector function is comparable to that of conventional CAR-T cells that directly target BCMA-positive tumor cells, but the activity of conventional CAR-T cells does not depend on the anti-BCMA/anti-P329G bispecific antibodies.

The in vitro experiments of the present invention show that only in the presence of anti-BCMA/anti-P329G bispecific antibodies, the activity of CAR-T cells containing P329G mutations in the extracellular domain can be "turned on", producing effector functions, identifying and killing BCMA-positive tumor cells. It is expected that by adjusting the dose and dosing interval of the anti-BCMA/anti-P329G bispecific antibody, the degree of in vivo expansion of CAR-T cells containing P329G mutations in the extracellular domain and the intensity of their anti-tumor activity can be regulated. In view of the fact that BCMA transmits tumor-promoting signals after binding to its ligand, the anti-BCMA part of the anti-BCMA/anti-P329G bispecific antibody can exert an anti-tumor effect by blocking this signal. Therefore, the anti-BCMA/anti-P329G bispecific antibody of the present invention and the CAR-T cells containing P329G mutations in the extracellular domain can also produce a synergistic anti-tumor effect.

In the seventh aspect, the present invention provides the use of the drug combination of the present invention for treating a BCMA-related disease in a subject, comprising administering to the subject a therapeutically effective amount of the drug combination defined in the aforementioned sixth aspect, wherein the BCMA-related disease is, for example, a cancer that expresses or overexpresses BCMA.

In an eighth aspect, the present invention provides use of the pharmaceutical combination of the present invention in the preparation of a medicament for treating a disease associated with BCMA, such as a cancer that expresses or overexpresses BCMA.

In a ninth aspect, the present invention provides a method for treating a disease associated with BCMA, the method comprising administering to a subject a therapeutically effective amount of the pharmaceutical combination of the present invention.

In a tenth aspect, the present invention provides a drug complex, which is a drug complex comprising

(i) immune effector cells (e.g., T cells, NK cells) expressing the CAR polypeptide described in the third aspect of the present invention; and

(ii) The anti-BCMA/anti-P329G bispecific antibody according to the first aspect of the present invention

A complex generated by binding of the P329G mutation contained in the extracellular domain of the CAR polypeptide to the anti-P329G of the anti-BCMA/anti-P329G bispecific antibody;

For example, the immune effector cells are T cells and NK cells expressing the CAR polypeptide described in the third aspect of the present invention prepared from autologous T cells, NK cells or allogeneic T cells and NK cells. For example, the immune effector cells are T cells and NK cells expressing the CAR polypeptide described in the third aspect of the present invention prepared from T cells and NK cells separated from human PBMC.

In an eleventh aspect, the present invention also provides use of the drug complex for treating a BCMA-related disease in a subject. Preferably, the BCMA-related disease is, for example, a cancer that expresses or overexpresses BCMA.

In a twelfth aspect, the present invention provides use of the drug complex of the present invention in the preparation of a medicament for treating a disease associated with BCMA, wherein the disease associated with BCMA is, for example, a cancer that expresses or overexpresses BCMA.

BRIEF DESCRIPTION OF THE DRAWINGS

The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show the currently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

Figure 1A, Figure 1B and Figure 1C respectively show schematic diagrams of three forms of anti-BCMA/anti-P329G bispecific antibodies recruiting T cells transduced by chimeric antigen receptors (CARs). The anti-BCMA/anti-P329G bispecific antibody has a BCMA tumor antigen targeting portion and a portion that specifically recognizes and binds to the P329G mutation present on the surface of CAR-T cells (also referred to herein as "anti-P329G" of the anti-BCMA/anti-P329G bispecific antibody); the CAR-T cell extracellular region contains the P329G mutation.

FIG2 shows schematic structural diagrams of three forms of anti-BCMA/anti-P329G bispecific antibody molecules (also referred to as molecule 1, molecule 2, and molecule 3).

Fig. 3 shows the schematic diagram of the structure of various chimeric antigen receptors (CAR) constructed by the present invention. The chimeric antigen receptor (CAR) comprises an extracellular domain, a G4S linker, a CD8 transmembrane domain (CD8TM), a 4-1BB intracellular co-stimulatory signaling domain, and a CD3ζ intracellular activation domain. The constructed chimeric antigen receptor (CAR) has different extracellular domains. In the figure, CH2ori represents that the extracellular domain of CAR-T cells is a wild-type IgG1 CH2 domain without any mutation or modification, and is sometimes referred to as CH2 without PG in the text (in the figure); CH2PG represents a single CH2 domain with a P329G mutation in the extracellular domain of CAR-T cells, and is also referred to as CH2P329G or simply CH2 in the text (in the figure); CH2PGAA represents a single CH2 domain with a P329G mutation in the extracellular domain of CAR-T cells and a truncated N-terminal portion (EAA removed), and is also referred to as CH2AA in the text (in the figure); CH2PGCH2PG represents that the extracellular domain of CAR-T cells contains two (G4S) ₄ linker connected CH2P329G domain, sometimes referred to as CH2CH2 in the text (in the figure); CH2PGCH3 represents the extracellular domain of CAR-T cells containing the CH2 domain with P329G mutation and the CH3 domain of normal IgG1, sometimes referred to as CH2CH3 in the text; CH2PGN297G represents the extracellular domain of CAR-T cells containing the CH2 domain with P329G and N297G mutations, sometimes referred to as CH2N297G in the text (in the figure).

Figure 4 shows a schematic diagram of the sequence structure of different chimeric antigen receptors (CAR) of the present invention. In the figure, "SP" represents a signal peptide; "G4S" represents a linker; "TMD" represents a transmembrane domain; "CSD" represents a costimulatory domain; "SSD" represents a stimulatory signaling domain. In the construct, according to the difference in the extracellular domain, it is divided into CH2ori (wild-type CH2 domain), CH2PG (CH2 domain containing P329G mutation), CH2PGAA (CH2 domain containing P329G mutation and truncated N-terminal part), CH2PGCH2PG (containing two CH2P329G domains connected by (G4S) 4 linkers), CH2PGCH3 (CH2 domain containing P329G mutation and CH3 domain of normal IgG1), CH2PGN297G (CH2 domain containing P329G, N297G mutation).

FIG5 shows the binding activity of the antibodies/half antibodies (antibody molecule 1, antibody molecule 3, and two half antibody molecules 2-1 and 2-2 constituting molecule 2) described in Example 1 to target cells expressing BCMA.

FIG6 shows the binding activity of two antibodies with different compositions described in Example 1 (molecule 1 and molecule 3) to Jurkat-NFAT cells expressing CH2P329GCAR.

Figure 7 shows the activation of Jurkat-NFAT cells expressing CH2P329G CAR in the presence of the antibodies/half antibodies (antibody molecule 1, antibody molecule 3 and two half antibody molecules 2-1 and 2-2 constituting molecule 2) described in Example 1 and H929 target cells.

Figure 8A shows the activation of Jurkat-NFAT cells expressing different CAR constructs of the present invention (extracellular domains are CH2, CH2CH2, CH2AA, CH2ori, CH2CH3 or CH2N297G, respectively) by molecule 1 in the presence of H929 target cells.

Figure 8B shows the activation of Jurkat-NFAT cells expressing different CAR constructs of the present invention (extracellular domains are CH2, CH2CH2, CH2AA, CH2ori, CH2CH3 or CH2N297G, respectively) by molecule 3 in the presence of H929 target cells.

FIG. 9 shows the expression of CH2ori CAR, CH2CH2 CAR, CH2CH3 CAR, and CH2CAR in primary T cells at day 10 after transduction.

Figure 10A shows the dynamic doubling of different CAR-T cells (extracellular domains are CH2ori, CH2CH2, CH2CH3, and CH2, respectively) monitored within 10 days after transduction.

Figure 10B shows the changes in diameter of different CAR-T cells (extracellular domains of which are CH2ori, CH2CH2, CH2CH3, and CH2, respectively) monitored within 10 days after transduction.

Figure 10C shows the changes in the survival rates of different CAR-T cells (extracellular domains are CH2ori, CH2CH2, CH2CH3, and CH2, respectively) monitored within 10 days after transduction.

FIG11A shows that under a fixed effector-target ratio (5:1), the killing effect of gradiently diluted molecule 1 on BCMA-positive HT1080 tumor cells (HT1080-BCMA) induced by CH2P329G CAR-T cells was detected by Xcelligence instrument.

FIG11B shows that under a fixed effector-target ratio (5:1), the killing effect of gradiently diluted molecule 3 induced by CH2P329G CAR-T cells on BCMA-positive HT1080 tumor cells (HT1080-BCMA) was detected by Xcelligence instrument.

Figure 12A shows that under the conditions of fixed antibody concentration (8 nM) and effector-target ratio (5:1), the killing effect of different CAR-T cells (extracellular domains of CH2ori, CH2CH2, CH2CH3, and CH2, respectively) induced by molecule 1 on BCMA-positive HT1080 tumor cells (HT1080-BCMA) was detected by Xcelligence instrument.

Figure 12B shows that under the conditions of fixed antibody concentration (8 nM) and effector-target ratio (5:1), the killing effect of different CAR-T cells (CH2ori, CH2CH2, CH2CH3, CH2) induced by molecule 3 on BCMA-positive HT1080 tumor cells (HT1080-BCMA) was detected by Xcelligence instrument.

Figure 12C shows that under the conditions of fixed antibody concentration (200nM) and effector-target ratio (5:1), the molecule 2 produced by the combination of half antibody molecule 2-1 and half antibody molecule 2-2 induced the killing effect of different CAR-T cells (CH2ori, CH2CH2, CH2CH3, CH2) on BCMA-positive HT1080 tumor cells (HT1080-BCMA) detected by Xcelligence instrument.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will be apparent from this specification and the accompanying drawings and from the appended claims.

I. Definitions

To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

As used herein, the term "and/or" means any one of the alternatives or two or more of the alternatives.

In this article, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the situation consisting of the elements, integers or steps mentioned. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.

The terms "BCMA" and "B cell maturation antigen" are used interchangeably and include variants, isoforms, species homologs, and analogs of human BCMA that have at least one identical epitope with BCMA (e.g., human BCMA). BCMA protein may also include fragments of BCMA, such as the extracellular domain of BCMA and fragments of the extracellular domain, such as fragments that retain the ability to bind to the anti-BCMA/anti-P329G bispecific antibodies of the present invention.

The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, half antibodies, and antibody fragments.

In some embodiments, the antibody of the present invention is an “anti-BCMA/anti-P329G bispecific antibody”, which comprises an anti-BCMA portion that specifically binds to BCMA (sometimes also referred to herein as “anti-BCMA”), and an anti-P329G portion that specifically binds to the P329G mutation (sometimes also referred to herein as “anti-P329G”).

"Antibody fragment" or "antigen-binding fragment" are used interchangeably herein and refer to a molecule different from an intact antibody, which comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fab, F(ab) ₂ , Fab', F(ab') ₂ , Fv, single-chain Fv, single-chain Fab, diabody, and half antibody.

The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are optionally connected continuously with the aid of a flexible short polypeptide linker, and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise indicated, as used herein, scFv can have VL variable regions and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL. In some embodiments, the antibody of the present invention is an "anti-P329G scFv" which includes a heavy chain variable region and a light chain variable region of anti-P329G.

"Complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" is a region in an antibody variable domain that is highly variable in sequence and forms structurally defined loops ("hypervariable loops") and/or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as CDR H1, CDR H2, and CDR H3, while the CDRs located within the antibody light chain variable domain are referred to as CDR L1, CDR L2, and CDR L3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al., (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (World Wide Web at imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-mentioned ways.

CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the CDRs exemplified in the present invention). In the present invention, when referring to antibody variable regions and specific CDR sequences (including heavy chain variable region residues), it refers to the numbering position according to the Kabat numbering system.

Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia or AbM can be replaced by conservative amino acid residues.

"Humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in the humanized antibody correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. The human IgG heavy chain Fc region is generally defined as a segment from the amino acid residue at the Cys226 or Pro230 position to the carboxyl terminus, and the lysine residue at position 447 at the C-terminus of the Fc region (according to the EU numbering system) may be present or absent. Thus, a full-length antibody composition may include an antibody group in which all K447 residues are eliminated, an antibody group in which no K447 residue is eliminated, or an antibody group in which antibodies with K447 residues and antibodies without K447 residues are mixed.

In certain embodiments, the Fc region of an immunoglobulin comprises two constant domains, namely, CH2 and CH3, and in other embodiments, the Fc region of an immunoglobulin comprises three constant domains, namely, CH2, CH3, and CH4.

“P329G” means that proline (P) at position P329 according to EU numbering is substituted with glycine (G) in the CH2 domain in the Fc region.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6 ^ed ., WH Freeman and Co. 91 pages (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity.

As used herein, the term "binding" or "specific binding" means that the binding is selective for an antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), SPR or biofilm interferometry or other conventional binding assays known in the art.

The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR/CD3 complex) to its corresponding ligand, which thereby mediates a signal transduction event, such as, but not limited to, signal transduction via a TCR/CD3 complex. Stimulation can mediate the expression of certain molecular changes, such as downregulation of TGF-β and/or reorganization of cytoskeletal structure, etc.

The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway. In one embodiment, the primary signal is initiated, for example, by the binding of the TCR/CD3 complex to the MHC molecule loaded with a peptide and leads to mediating T cell responses, including but not limited to proliferation, activation, differentiation, etc. In a specific CAR of the present invention, the intracellular signaling domain in any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, a primary signaling sequence of CD3ζ.

The term "CD3ζ" is defined as a protein provided by GenBank Accession No. BAG36664.1 or its equivalent, and "CD3ζ activation domain" is defined as amino acid residues from the cytoplasmic domain of the CD3ζ chain that are sufficient to functionally propagate the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of CD3ζ comprises residues 52 to residue 164 of GenBank Accession No. BAG36664.1 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) as their functional homologs. In one embodiment, the "CD3ζ activation domain" is a sequence provided in SEQ ID NO: 20 or a variant thereof.

The term "costimulatory molecule" refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a costimulatory response (such as but not limited to proliferation) of a cell. A costimulatory molecule is a cell surface molecule that contributes to an effective immune response except for an antigen receptor or its ligand. Costimulatory molecules include but are not limited to MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activation of NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28. In some embodiments, a "costimulatory molecule" is 4-1BB (i.e., CD137). A costimulatory domain refers to the intracellular portion of a costimulatory molecule.

The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence provided as GenBank Accession No. AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.). In one embodiment, the "4-1BB costimulatory domain" is a sequence provided as SEQ ID NO: 19 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).

The term "signaling pathway" refers to the biochemical relationships between various signaling molecules that play a role in propagating a signal from one part of a cell to another part of the cell.

The term "cytokine" is a generic term for proteins released by one cell population that act as intercellular mediators on another cell. Examples of such cytokines include lymphokines, monokines, interleukins (ILs), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factors, such as TNF-α or TNF-β; and other polypeptide factors, including gamma-interferon.

An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibodies of the invention are purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848: 79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An "isolated nucleic acid encoding an antibody of the invention" refers to one or more nucleic acid molecules that encode a chain of an antibody of the invention or a fragment thereof, including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

Calculation of sequence identity between sequences was performed as follows.

To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.

The comparison of sequences and calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm that has been integrated into the GAP program in the GCG software package (available at http://www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4).

Additionally or alternatively, one can further use the nucleic acid sequences and protein sequences described herein as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

The terms "amino acid change" and "amino acid modification" are used interchangeably and refer to the addition, deletion, substitution and other modifications of amino acids. Any combination of amino acid addition, deletion, substitution and other modifications can be performed, provided that the final polypeptide sequence has the desired properties. Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid changes can be produced using genetic or chemical methods known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. Methods for changing amino acid side chain groups by methods other than genetic engineering (such as chemical modification) may be useful. A variety of names may be used herein to represent the same amino acid change. For example, the substitution from proline at position 329 of the Fc domain to glycine can be represented as 329G, P329G, Pro329Gly, or simply referred to as "PG".

The terms "conservative sequence modifications" and "conservative sequence changes" refer to amino acid modifications or changes that do not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the interior of the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the functional assay described herein can be used to test the changed CAR.

The term "autologous" refers to any substance that is derived from the same individual into which the substance is later reintroduced.

The term "allogeneic" refers to any material that is derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species can be genetically dissimilar enough to interact antigenically.

The term "xenogeneic" refers to a graft derived from an animal of a different species.

As used herein, the term "apheresis" refers to an art-recognized extracorporeal method by which blood from a donor or patient is removed from the donor or patient and passed through a device that separates selected specific components and returns the remainder to the donor's or patient's circulation, e.g., by retransfusion. Thus, in the context of an "apheresis sample," refers to a sample obtained using apheresis.

The term "immune effector cell" refers to a cell that participates in an immune response, e.g., participates in promoting an immune effector reaction. Examples of immune effector cells include T cells, e.g., α/β T cells and γ/δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid derived phagocytes.

"Immune effector function", "immune effector response" or "immune effector reaction" refers to, for example, a function or response of an immune effector cell that enhances or promotes immune attack on a target cell. For example, an immune effector function or response refers to a T cell or NK cell property that promotes killing of target cells or inhibits growth or proliferation of target cells. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including the secretion of cytokines.

The term "T cell activation" refers to one or more cellular responses of T lymphocytes, particularly cytotoxic T lymphocytes, selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. The chimeric antigen receptor of the present invention is capable of inducing T cell activation. Suitable assays for measuring T cell activation are described in the Examples and are known in the art.

The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, and particularly includes self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, those from Oxford BioMedica. Gene delivery technology, LENTIMAX ^™ vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.

The term "BCMA-associated disease" refers to any condition caused by, exacerbated by, or otherwise associated with increased expression or activity of BCMA.

The terms "individual" or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

The terms "tumor" and "cancer" are used interchangeably herein and encompass both solid tumors and liquid tumors.

The terms "cancer" and "cancerous" refer to the physiological condition in mammals in which there is unregulated cell growth.

The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

"Tumor immune escape" refers to the process by which a tumor evades immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such escape is weakened and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

The term "half effective concentration ( _EC50 )" refers to the concentration of a drug, antibody or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time.

The term "fluorescence activated cell sorting" or "FACS" refers to a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time, based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting", 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS StarPlus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the Epics C from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).

The term "pharmaceutically acceptable excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, buffer, stabilizer, or the like, which is administered together with the active substance.

As used herein, "treat" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the drug combination of the present invention is used to delay the development of the disease or to slow the progression of the disease.

The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a drug box, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the anti-BCMA/anti-P329G bispecific antibody described in the first aspect of the present invention and (ii) the P329G CAR-T cells described in the fourth aspect of the present invention) are applied to the subject simultaneously, without specific time restrictions or at the same or different time intervals, sequentially as separate entities, wherein such administration provides effective treatment in the subject. The term "fixed combination" refers to the combination of the anti-BCMA/anti-P329G bispecific antibody and P329G CAR-T cells of the present invention, each of which is simultaneously applied to the patient in the form of a specific single dose. The term "non-fixed combination" means that the combination of the anti-BCMA/anti-P329G bispecific antibody and P329G CAR-T cells of the present invention is applied to the patient simultaneously, in parallel or sequentially as separate entities, without specific dosage and time restrictions, wherein such administration provides a therapeutically effective level of the drug combination of the present invention in the patient. In a preferred embodiment, the drug combination is a non-fixed combination.

The term "combination therapy" or "combination therapy" refers to the administration of two or more components to treat cancer as described in the present disclosure. Such administration includes the co-administration of these components in a substantially simultaneous manner. Alternatively, such administration includes the co-administration or separate administration or sequential administration of each active ingredient in a variety of or in separate containers (e.g., capsules, powders, and liquids). The powder and/or liquid can be reconstituted or diluted to the desired dose before administration. In some embodiments, administration also includes using the anti-BCMA/anti-P329G bispecific antibodies and P329G CAR-T cells of the present invention at approximately the same time, or in a sequential manner at different times. In either case, the treatment regimen will provide a beneficial effect of the drug combination in treating the disorders or conditions described herein.

The term "vector" as used herein when referring to nucleic acids refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are referred to herein as "expression vectors."

The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to parent cells in nucleic acid content, but may contain mutations. Included herein are mutant offspring with the same function or biological activity screened or selected in the initially transformed cells. Host cells are any type of cell system that can be used to produce antibody molecules of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, and also include cells inside transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

"Subject/patient sample" refers to a collection of cells, tissues or body fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as from fresh, frozen and/or preserved organ or tissue samples or biopsy samples or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time of pregnancy or development of the subject. Tissue samples may contain compounds that are naturally not mixed with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc. Examples of tumor samples include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage fluid, pleural fluid (pleural effusion), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.

The term "treat" or "treating" with reference to a disease means to alleviate the disease (ie, slow or arrest or reduce the development of the disease or at least one clinical symptom thereof), prevent or delay the onset or development or progression of the disease.

II. Molecular switch regulated chimeric antigen receptor (CAR) of the present invention

The present invention relates to a chimeric antigen receptor polypeptide that can specifically bind to an anti-P329G antibody or an antigen-binding fragment thereof. In some embodiments, the present invention relates to a chimeric antigen receptor polypeptide that can specifically bind to the anti-P329G portion of an anti-BCMA/anti-P329G bispecific antibody (also referred to as "anti-P329G").

The chimeric antigen receptor (CAR) polypeptide binding to anti-P329G of the present invention comprises:

(i) an extracellular domain, which is a mutant Fc domain or a fragment thereof or a combination of fragments thereof, wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G);

(ii) transmembrane domain;

(iii) a co-stimulatory domain; and

(iv) CD3ζ activation domain or a variant thereof having 1-10 amino acid modifications.

In some embodiments, the extracellular domain of the anti-P329G binding chimeric antigen receptor (CAR) polypeptide of the present invention can be any mutant Fc domain or a fragment thereof or a combination of fragments thereof, as long as the amino acid at the P329 position according to EU numbering is mutated to glycine (G).

The mutant Fc domain may be a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody, preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 antibody;

In some embodiments, the extracellular domain of the anti-P329G binding chimeric antigen receptor (CAR) polypeptide of the present invention is, for example, a single CH2 domain comprising a P329G mutation; a single CH2 domain comprising a P329G mutation and a truncation of 1-10 amino acid residues in the N-terminal portion; a CH2CH2 domain consisting of two CH2 domains comprising a P329G mutation; a CH2CH3 domain consisting of a CH2 domain containing a P329G mutation and a normal CH3 domain connected; or a single CH2 domain comprising P329G, N297G mutations.

Further, the extracellular domain can be connected to a signal peptide sequence at the N-terminus, for example, the signal peptide sequence shown in SEQ ID NO: 16, and the extracellular domain can be connected to a linker sequence as provided in SEQ ID NO: 17, a transmembrane region as provided in SEQ ID NO: 18, a co-stimulatory domain as provided in SEQ ID NO: 19, and an intracellular activation domain comprising SEQ ID NO: 20 or a variant thereof at the C-terminus, for example, wherein the respective domains are adjacent to each other and are in the same reading frame to form a single fusion protein.

In some embodiments, the extracellular domain comprises a P329G mutant Fc domain as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15, or a fragment thereof having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or a combination of fragments thereof.

In some embodiments, an exemplary CAR construct of the present invention comprises a signal peptide sequence, a P329G mutated Fc domain or a fragment thereof or a combination of fragments thereof, a linker sequence, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular activation domain.

In some embodiments, the present invention provides a recombinant nucleic acid construct comprising a nucleic acid molecule encoding the CAR of the present invention. The CAR construct encoding the present invention can be obtained using a recombinant method known in the art. Alternatively, the target nucleic acid can be produced synthetically rather than by a genetic recombination method.

The present invention includes retroviral and lentiviral vector constructs that express CARs that can be directly transduced into cells.

In some embodiments, the nucleic acid sequence of the CAR construct of the present invention is cloned into a lentiviral vector to generate a full-length CAR construct in a single coding frame, and the EF1α promoter is used for expression.

Those of ordinary skill in the art will appreciate that the CAR polypeptides of the present invention can also be modified so as to change in the amino acid sequence, but not in terms of the desired activity. For example, the CAR polypeptide may be subjected to additional nucleotide substitutions that result in amino acid substitutions at "non-essential" amino acid residues. For example, a non-essential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid fragment may be replaced with a structurally similar fragment that differs in the order and composition of the side chain family members, for example, a conservative substitution may be performed in which an amino acid residue is replaced with an amino acid residue having a similar side chain.

Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The transmembrane domain included in the CAR of the present invention is an anchored transmembrane domain, which is a component of a polypeptide chain that can be integrated into the cell membrane. The transmembrane domain can be fused with other extracellular and/or intracellular polypeptide domains, wherein these extracellular and/or intracellular polypeptide domains will also be restricted to the cell membrane. In the chimeric antigen receptor (CAR) polypeptide of the present invention, the transmembrane domain confers membrane attachment to the CAR polypeptide of the present invention. The CAR polypeptide of the present invention comprises at least one transmembrane domain, which may be derived from a natural source or a recombinant source, comprising dominant hydrophobic residues such as leucine and valine. In the case where the source is natural, the domain may be derived from a membrane-bound protein or a transmembrane protein such as CD28, CD8 (e.g., CD8α, CD8β) transmembrane domain. In one embodiment, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 18.

In some embodiments, the transmembrane domain in the CAR of the present invention is connected to the extracellular region of CAR (i.e., an Fc domain or a combination of its fragments or fragments comprising a P329G mutation) by means of a linker sequence. Glycine-serine doublets provide particularly suitable linker sequences. For example, in one embodiment, the linker comprises the amino acid sequence of GGGGS (SEQ ID NO: 17).

The cytoplasmic domain included in the CAR of the present invention includes an intracellular signaling domain. The intracellular signaling domain can activate at least one effector function of the immune cell introduced with the CAR of the present invention.

Examples of intracellular signaling domains for use in the CARs of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after combination with the extracellular domains, as well as any derivatives or variants of these sequences and any recombinant sequences with the same functional capabilities.

Considering that the signal generated by TCR alone is not enough to fully activate T cells, the CAR of the present invention is also designed to generate a costimulatory domain (CSD) capable of generating a costimulatory signal. The activation of T cells is mediated by two different cytoplasmic signaling sequences: those sequences (primary intracellular signaling domains) that initiate antigen-dependent primary activation by TCR and those sequences (secondary cytoplasmic domains, e.g., costimulatory domains) that act in an antigen-independent manner to provide costimulatory signals.

In one embodiment, the CAR of the present invention comprises a primary intracellular signaling domain, e.g., a primary signaling domain of CD3ζ, e.g., a CD3ζ activation domain set forth in SEQ ID NO:20.

The intracellular signaling domain in the CAR of the present invention also includes a secondary signaling domain (ie, a co-stimulatory domain). The co-stimulatory domain refers to the CAR portion of the intracellular domain comprising a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for lymphocytes to effectively respond to antigens in addition to antigen receptors or their ligands. In some embodiments, co-stimulatory molecules include but are not limited to CD28, 4-1BB (CD137), and the co-stimulatory effect caused by them enhances the proliferation, effector function and survival of human CAR-T cells in vitro and enhances the anti-tumor activity of human T cells in vivo.

Intracellular signaling sequences of the CAR of the present invention can be connected to each other in a random order or in a specified order. Optionally, short oligopeptide linkers or polypeptide linkers can form bonds between intracellular signaling sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, for example, alanine, glycine, can be used as a suitable linker.

In one embodiment, the intracellular signaling domain of the CAR of the present invention is designed to include the co-stimulatory domain of 4-1BB and the activation domain of CD3ζ.

III. Nucleic acid molecules encoding the CAR of the present invention, vectors, and cells expressing the CAR of the present invention

The present invention provides nucleic acid molecules encoding CAR constructs described herein.In one embodiment, the nucleic acid molecule is provided as a DNA construct.

The present invention also provides a vector inserted with a CAR construct of the present invention. By effectively connecting a nucleic acid encoding a CAR polypeptide to a promoter and incorporating the construct into an expression vector, expression of a natural or synthetic nucleic acid encoding CAR is achieved. The vector may be suitable for replication and integration in eukaryotic organisms. Common cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for regulating the expression of the desired nucleic acid sequence.

Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo. Numerous retroviral systems are known in the art. In some embodiments, a lentiviral vector is used.

Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenics and their propagation in daughter cells. Lentiviral vectors have the additional advantage of being superior to vectors derived from cancer-retroviruses (such as murine leukemia viruses) because they can transduce non-proliferative cells, such as hepatocytes. They also have additional low immunogenicity advantages. Retroviral vectors can also be, for example, γ retroviral vectors. γ retroviral vectors can, for example, include promoters, packaging signals (ψ), primer binding sites (PBS), one or more (e.g., two) long terminal repeats (LTR) and target transgenics, for example, genes encoding CAR. γ retroviral vectors can lack viral structural genes such as gag, pol and env.

An example of a promoter capable of expressing a CAR transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to effectively drive the expression of CAR from a transgene cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).

Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutive strong promoter sequence that can drive any polynucleotide sequence effectively connected thereto to express at a high level. However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, elongation factor-1α promoter, hemoglobin promoter and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.

In some embodiments, the present invention provides methods for expressing a CAR construct of the present invention in a mammalian immune effector cell (e.g., a mammalian T cell or a mammalian NK cell) and the immune effector cells produced thereby.

A cell source (e.g., immune effector cells, e.g., T cells or NK cells) is obtained from a subject. The term "subject" is intended to include living organisms (e.g., mammals) that can stimulate an immune response. T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors.

Any technique known to those skilled in the art (such as Ficoll ^TM separation) can be used to obtain T cells from blood components collected from a subject. In a preferred aspect, cells from individual circulating blood are obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and to place cells in a suitable buffer or culture medium for subsequent processing steps. In one aspect of the invention, cells are washed with phosphate buffered saline (PBS).

Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, by using beads conjugated with anti-CD3/anti-CD28 (e.g. In some embodiments, the time period is about 30 minutes to 36 hours or longer. Longer incubation time can be used to separate T cells in any case where there is a small amount of T cells, such as for separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. In addition, the efficiency of capturing CD8+T cells can be increased using longer incubation time. Therefore, by simply shortening or extending the time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, T cell subsets can be selected preferentially at the beginning of culture or at other time points during the culture process.

Enrichment of T cell populations can be accomplished by a negative selection process using a combination of antibodies. One approach is to sort and/or select cells using negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells.

In some embodiments, immune effector cell can be an allogeneic immune effector cell, for example, a T cell or a NK cell. For example, the cell can be an allogeneic T cell, for example, an allogeneic T cell lacking a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (for example, HLA class I and/or HLA class II) expression.

A T cell lacking a functional TCR can, for example, be engineered so that it does not express any functional TCR on its surface; engineered so that it does not express one or more subunits that make up a functional TCR (e.g., engineered so that it does not express or shows reduced expression of TCRα, TCRβ, TCRγ, TCRδ, TCRε and/or TCRζ); or engineered so that it produces very few functional TCRs on its surface.

The T cells described herein can be engineered, for example, so that it does not express functional HLA on its surface. For example, the T cells described herein can be engineered so that the cell surface expression of HLA (e.g., HLA class I and/or HLA class II) is downregulated. In some aspects, downregulation of HLA can be achieved by reducing or eliminating beta-2 microglobulin (B2M) expression.

In some embodiments, the T cell may lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II.

In some embodiments, the immune effector cell can be an autologous immune effector cell, e.g., a T cell or a NK cell, e.g., the cell can be an autologous T cell, e.g., a T cell isolated from human PBMC.

In one embodiment, cells transduced with a nucleic acid encoding a CAR described herein are proliferated, for example, the cells are proliferated in culture for 2 hours to about 14 days.

The CAR-expressing immune effector cells obtained after in vitro proliferation can be tested for effector function as described in the examples.

IV. Anti-BCMA/Anti-P329G Bispecific Antibodies

The present invention provides an anti-BCMA/anti-P329G bispecific antibody that specifically binds to BCMA and specifically binds to the P329G mutation, comprising an anti-BCMA portion that specifically binds to BCMA and an anti-P329G portion that specifically binds to the P329G mutation.

In one embodiment, the anti-BCMA/anti-P329G bispecific antibody comprises two heavy chains and two light chains, and has a F(ab) ₂ that specifically binds to BCMA, a heavy chain variable region that specifically binds to an anti-P329G mutation that is located at the C-terminus of the CH3 domain of one heavy chain, and a light chain variable region that specifically binds to an anti-P329G mutation that is located at the C-terminus of the CH3 domain of the other heavy chain.

In one embodiment, the anti-BCMA/anti-P329G bispecific antibody comprises two heavy chains and two light chains, and has two half antibodies consisting of one heavy chain and one light chain, wherein one half antibody has an N-terminal Fab that specifically binds to BCMA and an anti-P329G heavy chain variable region that specifically binds to the P329G mutation located between the C-terminus of the heavy chain Fab and the N-terminus of the CH2 domain, and the other half antibody has an N-terminal Fab that specifically binds to BCMA and a light chain variable region that specifically binds to the P329G mutation located between the C-terminus of the heavy chain Fab and the N-terminus of the CH2 domain.

In one embodiment, the anti-BCMA/anti-P329G bispecific antibody comprises two heavy chains and two light chains, and has a F(ab) ₂ that specifically binds to BCMA and an anti-P329G scFv that specifically binds to the P329G mutation, wherein the C-terminus of the CH3 domain of one heavy chain is connected to the N-terminus of the anti-P329G scFv.

In some embodiments, the anti-BCMA portion that specifically binds to BCMA in the anti-BCMA/anti-P329G bispecific antibody is in the form of Fab, Fab', F(ab') ₂ , Fv, single-chain Fv, or single-chain Fab, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDRH1 shown in the amino acid sequence SSSYYWT (SEQ ID NO: 25) according to Kabat numbering, or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR H2 shown in the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR H3 shown in the amino acid sequence DRGDQILDV (SEQ ID NO: 27), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises the CDR L1, or a variant of said CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR L2 shown in the amino acid sequence AASSLQS (SEQ ID NO: 29), or a variant of said CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR L3 shown in the amino acid sequence QQKYFDIT (SEQ ID NO: 30), or a variant of said CDR L3 with no more than 2 amino acid changes or no more than 1 amino acid change;

The amino acid change is an addition, deletion or substitution of an amino acid.

In one embodiment, the anti-BCMA scFv domain optionally comprises a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4. The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

In some embodiments, the anti-BCMA/anti-P329G bispecific antibodies of the invention bind to mammalian BCMA, such as human, cynomolgus monkey, mouse, rat, and rabbit BCMA.

In some embodiments, the anti-BCMA/anti-P329G bispecific antibody of the present invention comprises a heavy chain variable region and a light chain variable region that specifically binds to BCMA, wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 23, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 24, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the heavy chain variable region is the sequence of SEQ ID NO: 23, and the light chain variable region is the sequence of SEQ ID NO: 24. The amino acid changes in the sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are preferably amino acid substitutions, more preferably conservative amino acid substitutions. Preferably, the amino acid changes do not occur in the CDR regions.

In some embodiments, the anti-P329G portion that specifically binds to the P329G mutation in the anti-BCMA/anti-P329G bispecific antibody is in the form of Fab, Fab', Fv, single-chain Fv, or single-chain Fab, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain complementary determining region CDR H1 shown in the amino acid sequence RYWMN (SEQ ID NO: 31) according to Kabat numbering, or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; a CDR H2 shown in the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 32), or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and a CDR H3 shown in the amino acid sequence PYDYGAWFAS (SEQ ID NO: 33), or a variant of the CDR H3 with no more than 2 amino acid changes or no more than 1 amino acid change; the light chain variable region comprises the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 34) according to Kabat numbering. NO: 34), or a variant of said CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR L2 with the amino acid sequence GTNCRAP (SEQ ID NO: 35), or a variant of said CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and CDR L3 with the amino acid sequence ALWYSNHWV (SEQ ID NO: 36), or a variant of said CDR L3 with no more than 2 amino acid changes or no more than 1 amino acid change;

The amino acid change is an addition, deletion or substitution of an amino acid.

In one embodiment, the anti-P329G scFv domain optionally comprises a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5 or 6, preferably 3 or 4. The light chain variable region and the heavy chain variable region of the scFv can be, for example, in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

In some embodiments, the anti-BCMA/anti-P329G bispecific antibody of the present invention comprises a heavy chain variable region and a light chain variable region that specifically binds to the P329G mutation, wherein the heavy chain variable region comprises a sequence of SEQ ID NO: 21, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain variable region comprises a sequence of SEQ ID NO: 22, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the heavy chain variable region is the sequence of SEQ ID NO: 21, and the light chain variable region is the sequence of SEQ ID NO: 22. The amino acid changes in the sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are preferably amino acid substitutions, more preferably conservative amino acid substitutions. Preferably, the amino acid changes do not occur in the CDR regions.

In some embodiments, the anti-BCMA/anti-P329G bispecific antibody of the present invention is an IgG1 or IgG4 antibody; more preferably, it is an IgG1 antibody, for example, a human IgG1 antibody.

In some embodiments, the Fc domains of the two heavy chains of the anti-BCMA/anti-P329G bispecific antibodies of the present invention respectively contain a protrusion ("knob") or a cavity ("hole"), and the protrusion or cavity in the Fc domain of one heavy chain can be placed in the cavity or protrusion in the Fc domain of the other heavy chain, respectively, so that the two heavy chains form a "knob-in-hole" stable association with each other. In one embodiment, one of the two heavy chains comprises an amino acid substitution T366W, and the other of the two heavy chains comprises amino acid substitutions T366S, L368A, and Y407V (EU numbering). Thus, the protrusion in one chain can be placed in the cavity in the other chain, promoting the correct pairing of the two heavy chains of the anti-BCMA/anti-P329G bispecific antibodies.

In some embodiments, the present invention provides nucleic acids encoding any of the above anti-BCMA/anti-P329G bispecific antibodies or any of their chains. In one embodiment, a vector comprising the nucleic acid is provided. In one embodiment, the vector is an expression vector. In one embodiment, a host cell comprising the nucleic acid or the vector is provided. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells), or other cells suitable for preparing antibodies or their antigen-binding fragments. In another embodiment, the host cell is prokaryotic.

For example, the nucleic acid of the present invention comprises a nucleic acid encoding an anti-BCMA/anti-P329G bispecific antibody of the present invention. In some embodiments, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. The vector includes but is not limited to a virus, a plasmid, a cosmid, a λ phage, or a yeast artificial chromosome (YAC). In one embodiment, the vector is a pcDNA3.4 expression vector.

Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultivated in a culture medium and screened for suitable activity. Methods and conditions for culturing the transfected cells produced and for recovering the antibody molecules produced are known to those skilled in the art and can be based on this specification and methods known in the prior art, depending on the specific expression vector and mammalian host cell used. Variation or optimization.

In addition, one or more markers of the host cell that has been transfected can be selected by introducing, and the cell that has stably incorporated DNA into its chromosome can be selected. The marker can, for example, provide prototrophy, biocidal resistance (for example, antibiotic) or heavy metal (such as copper) resistance etc. to the auxotrophic host. The selectable marker gene can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be needed so that the best synthetic mRNA can be obtained. These elements can include splicing signals, as well as transcription promoters, enhancers and termination signals.

In one embodiment, a host cell comprising a polynucleotide of the present invention is provided. In some embodiments, a host cell comprising an expression vector of the present invention is provided. In some embodiments, the host cell is selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies. Suitable host cells include prokaryotic microorganisms, such as Escherichia coli. The host cell can also be a eukaryotic microorganism such as a filamentous fungus or yeast, or various eukaryotic cells, such as insect cells, etc. Vertebrate cells can also be used as hosts. For example, a mammalian cell line modified to be suitable for suspension growth can be used. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (HEK293 or 293F cells), 293 cells, baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), Chinese hamster ovary cells (CHO cells), CHO-S cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63 and Sp2/0, etc. For a review of mammalian host cell lines suitable for producing proteins, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In a preferred embodiment, the host cell is a CHO cell or a HEK293 cell.

In one embodiment, the present invention provides a method for preparing an anti-BCMA/anti-P329G bispecific antibody, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the anti-BCMA/anti-P329G bispecific antibody or an expression vector comprising the nucleic acid under conditions suitable for expressing the nucleic acid encoding the anti-BCMA/anti-P329G bispecific antibody, and optionally isolating the anti-BCMA/anti-P329G bispecific antibody. In a certain embodiment, the method further comprises recovering the anti-BCMA/anti-P329G bispecific antibody from the host cell (or host cell culture medium).

In one embodiment, the present invention prepares an anti-BCMA/anti-P329G bispecific antibody as two half antibodies thereof, respectively, and then obtains a complete anti-BCMA/anti-P329G bispecific antibody by combining the two half antibodies. The preparation method comprises culturing a host cell introduced with an expression vector encoding a nucleic acid of one of the two half antibodies of the anti-BCMA/anti-P329G bispecific antibody of the present invention under conditions suitable for expressing nucleic acids encoding the two half antibodies in the anti-BCMA/anti-P329G bispecific antibody of the present invention, isolating one half antibody of the anti-BCMA/anti-P329G bispecific antibody, and culturing a host cell introduced with an expression vector encoding a nucleic acid of the other half antibody of the two half antibodies of the anti-BCMA/anti-P329G bispecific antibody of the present invention, isolating the other half antibody of the anti-BCMA/anti-P329G bispecific antibody, and optionally the method further comprises recovering the two half antibodies from the host cell; the combination of the two half antibodies constitutes the anti-BCMA/anti-P329G bispecific antibody.

The anti-BCMA/anti-P329G bispecific antibodies of the present invention prepared as described herein or the two half antibodies constituting the same can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these are obvious to those skilled in the art. The purity of the anti-BCMA/anti-P329G bispecific antibodies of the present invention or the two half antibodies constituting the same can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

The anti-BCMA/anti-P329G bispecific antibodies provided herein or the two half antibodies constituting them can be identified, screened or characterized by various assays known in the art for their physical/chemical properties and/or biological activity. On the one hand, the anti-BCMA/anti-P329G bispecific antibodies of the present invention or the two half antibodies constituting them are tested for their antigen binding activity, for example, by known methods such as FACS, ELISA or Western blotting, etc. The binding to BCMA can be determined using methods known in the art, and exemplary methods are disclosed herein. In some embodiments, the binding of the anti-BCMA/anti-P329G bispecific antibodies of the present invention or the two half antibodies constituting them to cell surface BCMA (eg, human BCMA) is determined using FACS.

The present invention also provides an assay for identifying an anti-BCMA/anti-P329G bispecific antibody or two half antibodies constituting the same having biological activity.

Cells used in any of the above in vitro assays include cell lines that naturally express BCMA or are engineered to express BCMA. The cell lines engineered to express BCMA are cell lines that do not normally express BCMA but express BCMA after transfection of BCMA-encoding DNA into the cells.

V. Pharmaceutical Combinations of the Present Invention

For optimizing the safety and efficacy of CAR therapy, the molecular switch regulated chimeric antigen receptor of the present invention is a regulatable CAR that can control the activity of CAR. The present invention uses anti-BCMA/anti-P329G bispecific antibodies as a safety switch in the CAR treatment of the present invention. In the absence of the anti-BCMA/anti-P329G bispecific antibody, the CAR activity of the present invention is turned off; in the presence of the anti-BCMA/anti-P329G bispecific antibody, the CAR activity of the present invention is turned on; thus, the opening and closing of the CAR molecule activity of the present invention is regulated by the anti-BCMA/anti-P329G bispecific antibody.

In some embodiments, the present invention provides a drug combination, which includes (i) immune effector cells (e.g., T cells, NK cells) expressing the molecular switch regulated CAR polypeptides of the present invention; and (ii) anti-BCMA/anti-P329G bispecific antibodies specifically binding to BCMA and specifically binding to P329G mutations. For example, the immune effector cell is a T cell expressing the molecular switch regulated CAR polypeptide of the present invention prepared from autologous T cells or allogeneic T cells, for example, the immune effector cell is a T cell expressing the molecular switch regulated CAR polypeptide of the present invention prepared from T cells separated from human PBMC. In some embodiments, the anti-BCMA/anti-P329G bispecific antibody includes:

i) two light chains as set forth in SEQ ID NO: 2 and two heavy chains as set forth in SEQ ID NO: 1 and SEQ ID NO: 3, respectively, or sequences at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to any of said sequences; or

ii) two light chains as set forth in SEQ ID NO: 2 and two heavy chains as set forth in SEQ ID NO: 4 and SEQ ID NO: 5, respectively, or sequences at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to any of said sequences; or

iii) two light chains as set forth in SEQ ID NO:2 and two heavy chains as set forth in SEQ ID NO:6 and SEQ ID NO:7, respectively, or sequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical to any of said sequences.

In some embodiments, the present invention provides a drug combination comprising (i) a nucleic acid molecule encoding a molecular switch-regulated CAR polypeptide of the present invention or a vector comprising the nucleic acid component; and (ii) an anti-BCMA/anti-P329G bispecific antibody that specifically binds to BCMA and specifically binds to the P329G mutation.

In some embodiments, the pharmaceutical combination of the present invention optionally further comprises a pharmaceutically acceptable excipient of a suitable formulation. For example, (ii) in the pharmaceutical combination can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A.).

In some embodiments, the pharmaceutical combination of the present invention is used to treat a disease associated with BCMA, such as a cancer that expresses or overexpresses BCMA.

VI. Uses of the pharmaceutical combination of the present invention and methods of treatment using the pharmaceutical combination of the present invention

The present invention provides the aforementioned pharmaceutical combination of the present invention for use in treating a disease associated with BCMA in a subject.

In one embodiment, the pharmaceutical combination of the present invention is used to treat a cancer that expresses or overexpresses BCMA in a subject and is capable of reducing the severity of at least one symptom or indication of the cancer or inhibiting the growth of cancer cells.

The present invention provides a method for treating a BCMA-related disease (eg, a cancer that expresses or overexpresses BCMA) in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical combination of the present invention.

The present invention provides use of the aforementioned drug combination of the present invention in the preparation of a medicament for treating a disease associated with BCMA (eg, a cancer expressing or overexpressing BCMA).

The pharmaceutical combinations of the invention may also be administered to individuals whose cancer has been treated with one or more prior therapies but has subsequently relapsed or metastasized.

In some embodiments, (i) immune effector cells (e.g., T cells, NK cells) expressing the molecular switch regulated CAR polypeptides of the present invention and (ii) anti-BCMA/anti-P329G bispecific antibodies specifically binding to BCMA and specifically binding to P329G mutations in the drug combination of the present invention are used for parenteral, percutaneous, intracavitary, intra-arterial, intravenous, intrathecal administration, or directly injected into tissues or tumors. In some embodiments, (ii) anti-BCMA/anti-P329G bispecific antibodies specifically binding to BCMA and specifically binding to P329G mutations in the drug combination of the present invention are administered before, simultaneously with, or after (i) immune effector cells (e.g., T cells, NK cells) expressing the molecular switch regulated CAR polypeptides of the present invention.

In some embodiments, (i) the immune effector cells expressing the molecular switch-regulated CAR polypeptide of the present invention in the drug combination of the present invention are T cells expressing the CAR polypeptide of the present invention prepared from autologous T cells or allogeneic T cells; (ii) the anti-BCMA/anti-P329G bispecific antibody in the drug combination of the present invention is any anti-BCMA/anti-P329G bispecific antibody that can specifically bind to BCMA and specifically bind to the P329G mutation.

Drug combination of the present invention can be applied to the subject at a suitable dosage. The dosage regimen will be determined by the attending physician and clinical factors. As known in the medical field, the dosage for any one patient depends on many factors, including the patient's body weight, body surface area, age, specific compound to be administered, sex, administration time and route, general health status and other drugs to be administered in parallel.

In some embodiments, administering the drug combination of the present invention to an individual suffering from cancer results in the complete disappearance of a tumor. In some embodiments, administering the drug combination of the present invention to an individual suffering from cancer results in a reduction of tumor cells or tumor size by at least 85% or more. The reduction of a tumor can be measured by any method known in the art, such as X-ray, positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), cytology, histology, or molecular genetic analysis.

In some embodiments, the drug combination of the present invention can reduce the "on-target/off tumor" toxicity of CAR-T cells.

The various embodiments/technical solutions described in the present invention and the features in each embodiment/technical solution should be understood to be able to be combined with each other arbitrarily, and the various solutions obtained by these combinations are included in the scope of the present invention, just as the solutions obtained by these combinations are specifically and one by one listed in this document, unless the context clearly shows otherwise.

The following examples are described to assist understanding of the present invention. The examples are not intended to and should not be interpreted in any way as limiting the scope of protection of the present invention.

Example

Example 1. Anti-BCMA/anti-P329G bispecific antibody and its preparation

In this example, three compositions of anti-BCMA/anti-P329G bispecific antibodies were prepared.

Figure 1A is a schematic diagram of the first compositional form of anti-BCMA/anti-P329G bispecific antibody recruiting P329G CAR-T cells. The first compositional form of anti-BCMA/anti-P329G bispecific antibody is also referred to as antibody molecule 1 or molecule 1, which comprises an F(ab) ₂ end targeting the BCMA antigen on the surface of tumor cells, and an anti-P329G VH domain and VL domain for the P329G mutation in the IgG1 CH2 domain, wherein the anti-P329G VH domain is connected to the C-terminus of one of the IgG1 heavy chain CH3 domains, and the anti-P329G VL domain is connected to the C-terminus of the CH3 domain of another IgG1 heavy chain. The extracellular region of the CAR expressed on the surface of CAR-T cells contains a P329G mutation in the CH2 domain. The anti-P329G in the anti-BCMA/anti-P329G bispecific antibody recruits and activates the activity of P329G CAR-T cells by binding to the P329G mutation.

Figure 1B is a schematic diagram of the second composition form of anti-BCMA/anti-P329G bispecific antibody recruiting CAR-T cells. The second composition form of anti-BCMA/anti-P329G bispecific antibody is also referred to as molecule 2, which consists of two half antibodies, respectively called half antibody 2-1 and half antibody 2-2, which can be expressed and synthesized by two different cell lines. Half antibody 2-1 includes a Fab end targeting the BCMA antigen on the surface of tumor cells, and a VH domain of anti-P329G for the P329G mutation of the IgG1 CH2 domain at the C-terminus of the heavy chain Fab and the N-terminus of the CH2 domain; half antibody 2-2 includes a Fab end targeting the BCMA antigen on the surface of tumor cells, and a VL domain of anti-P329G for the P329G mutation of the IgG1 CH2 domain at the C-terminus of the heavy chain Fab and the N-terminus of the CH2 domain. Both half antibody 2-1 and half antibody 2-2 can target the tumor antigen BCMA. After half antibody 2-1 and half antibody 2-2 bind to the tumor antigen BCMA, the anti-P329G VH domain in half antibody 2-1 and the anti-P329G VL domain in half antibody 2-2 are close to form a complete anti-P329G targeting the P329G mutation. Thus, by binding to the P329G mutation on the surface of CAR-T cells, the activity of P329G CAR-T cells is recruited and activated.

Figure 1C is a schematic diagram of the third composition form of anti-BCMA/anti-P329G bispecific antibody recruiting CAR-T cells. The third composition form of anti-BCMA/anti-P329G bispecific antibody is also referred to as molecule 3, which includes an F(ab) ₂ end targeting the BCMA antigen on the surface of tumor cells, and an anti-P329G scFv for the P329G mutation in the IgG1 CH2 domain, wherein the C-terminus of the CH3 domain of one heavy chain is connected to the N-terminus of the anti-P329G scFv. The extracellular region of the CAR expressed on the surface of CAR-T cells contains a P329G mutation. The anti-P329G in the anti-BCMA/anti-P329G bispecific antibody recruits and activates the activity of P329G CAR-T cells by binding to the P329G mutation.

FIG. 2 shows three different compositions of anti-BCMA/anti-P329G bispecific antibodies.

The anti-BCMA/anti-P329G bispecific antibody molecule 1 (also referred to herein as molecule 1) comprises a heavy chain 1.1 (the sequence is shown in SEQ ID NO: 1), a heavy chain 1.2 (the sequence is shown in SEQ ID NO: 3), and a light chain (the sequence is shown in SEQ ID NO: 2).

The anti-BCMA/anti-P329G bispecific antibody molecule 2 (also referred to herein as molecule 2) is composed of a half antibody molecule 2-1 and a half antibody molecule 2-2, wherein molecule 2-1 comprises a heavy chain 2.1 (sequence as shown in SEQ ID NO: 4) and a light chain (sequence as shown in SEQ ID NO: 2); molecule 2-2 comprises a heavy chain 2.2 (sequence as shown in SEQ ID NO: 5) and a light chain (sequence as shown in SEQ ID NO: 2).

The anti-BCMA/anti-P329G bispecific antibody molecule 3 (also referred to herein as molecule 3) comprises a heavy chain 3.1 (the sequence of which is shown in SEQ ID NO: 6), a heavy chain 3.2 (the sequence of which is shown in SEQ ID NO: 7), and a light chain (the sequence of which is shown in SEQ ID NO: 2).

The above-mentioned anti-BCMA/anti-P329G bispecific antibodies with different structures can be prepared as follows: the coding nucleic acid sequence of the above-mentioned anti-BCMA/anti-P329G bispecific antibody is synthesized by whole gene. The nucleic acid sequence encoding the anti-BCMA/anti-P329G bispecific antibody is cloned into the pcDNA3.4 expression vector (purchased from Shanghai Baiying) respectively, and the obtained pcDNA3.4 expression vector containing the heavy chain and the pcDNA3.4 expression vector containing the light chain are co-transfected into HEK293 cells by PEI at a mass ratio of 2:3, and the culture supernatant is collected after culturing for 5 to 7 days to obtain the anti-BCMA/anti-P329G bispecific antibody.

Molecule 2 can also be prepared as follows. Molecule 2 consists of two half-antibody parts (heavy chain plus light chain) called molecule 2-1 and molecule 2-2. Molecule 2-1 and molecule 2-2 are expressed in two independent HEK293T cells and obtained separately. The specific operation is: the pcDNA3.4 expression vector containing heavy chain 2.1 and the pcDNA3.4 expression vector containing light chain (purchased from Shanghai Boying) are co-transfected into HEK293 cells by PEI at a mass ratio of 2:3; similarly, the pcDNA3.4 expression vector containing heavy chain 2.2 and the pcDNA3.4 expression vector containing light chain (purchased from Shanghai Boying) are co-transfected into HEK293 cells by PEI at a mass ratio of 2:3; after culturing two independent HEK293T cells for 5 to 7 days, the culture supernatant is collected to obtain half-antibody molecule 2-1 and half-antibody molecule 2-2 respectively.

The supernatant medium containing antibody/half antibody was purified by a Protein A column and then dialyzed with PBS. The concentration was detected by reading the absorbance at 280 nm using a NanoDrop instrument, and the sample purity was detected by SDS-PAGE and SEC-HPLC methods. Antibody molecule 1, antibody molecule 3, half antibody molecule 2-1 and half antibody molecule 2-2 were obtained.

The control anti-P329G IgG comprises a heavy chain (sequence as shown in SEQ ID NO: 8) and a light chain (sequence as shown in SEQ ID NO: 9). The pcDNA3.4 expression vectors (purchased from Shanghai Bio-Invitrogen) containing the control anti-P329G IgG light chain and heavy chain, respectively, were co-transfected into HEK293 cells by PEI at a mass ratio of 2:3. After culturing for 5 to 7 days, the culture supernatant was collected and further purified (the purification method was the same as above) to obtain the control anti-P329G IgG antibody.

Example 2. Preparation of P329G CAR construct and P329G CAR expression vector

Figure 3 shows a schematic diagram of the structure of a P329G chimeric antigen receptor (CAR) in different composition forms. Figure 4 shows a schematic diagram of the sequence structure of different chimeric antigen receptor (CAR) constructs. All CAR constructs have an Fc region CH2 domain expressed on the cell surface.

According to the different extracellular domains of CAR, CAR is named as follows:

- CH2ori or CH2 no PG (SEQ ID NO: 10), i.e., wild-type IgG1 CH2 domain without any mutation or modification;

- CH2PG or CH2 P329G or CH2 (SEQ ID NO: 11), i.e., a single CH2 domain comprising a P329G mutation in the CH2 domain of the Fc region;

- CH2PGAA or CH2AA (SEQ ID NO: 12), i.e., a single CH2 domain containing a P329G mutation and a truncated N-terminal portion;

- CH2PGCH2PG or CH2CH2 (SEQ ID NO: 13), i.e., comprising two CH2P329G domains connected by a (G4S) ₄ linker;

- CH2PGCH3 or CH2CH3 (SEQ ID NO: 14), i.e., a CH2 domain containing a P329G mutation and a CH3 domain of normal IgG1;

- CH2PGN297G or CH2N297G (SEQ ID NO: 15), i.e., a CH2 domain containing P329G, N297G mutations.

The different extracellular domains are all connected to the CD8 transmembrane domain (CD8TM), the 4-1BB intracellular co-stimulatory signaling domain and the CD3ζ intracellular activation domain through a G4S linker.

The preparation process of the expression vectors of the above-mentioned different CAR molecules is as follows: the above-mentioned DNA fragment is inserted into the downstream of the EF1α promoter of the pRK lentiviral expression vector (which is modified by replacing the promoter and the resistance gene of the pRRLSIN.cPPT.PGK-GFP.WPRE vector (Addgene, 12252, purchased from BioWind)), and the EGFP sequence in the original vector is replaced to obtain different P329GCAR expression vectors.

Example 3. Detection of Binding Activity of Anti-BCMA/Anti-P329G Bispecific Antibody to BCMA Antigen

The positive multiple myeloma cell line H929 expressing BCMA (purchased from Nanjing Kebai Biotechnology Co., Ltd., CBP60243) was cultured. An appropriate amount of H929 tumor cells in the logarithmic growth phase were taken, washed twice with FACS buffer, and the antibody molecule 1, half antibody molecule 2-1, half antibody molecule 2-2, and antibody molecule 3 prepared in Example 1 were added with gradient dilution, stained at 4°C for 30 minutes, washed twice, and then the H929 tumor cells were resuspended with FACS buffer, and Biotin-Protein L (AcroBiosystems, RPL-P814R) was added, and incubated at 4°C for 30 minutes; after washing twice with FACS buffer, APC-Streptavidin (Biolegend, 405207) was added, and stained at 4°C for 30 to 45 minutes; after washing twice with FACS buffer, the tumor cells were resuspended with FACS buffer and detected by flow cytometry. The antibody/half antibody concentration was used as the X-axis and the proportion of the APC-positive cell population was used as the Y-axis to draw a graph and calculate the EC50 of antibody binding.

Figure 5 shows the binding activity of different antibodies/half antibodies to the BCMA-expressing positive multiple myeloma cell line H929 at different concentrations. The results show that antibody molecule 1, half antibody molecule 2-1, half antibody molecule 2-2, and antibody molecule 3 can bind to BCMA-expressing positive tumor cells and show antibody/half antibody molecule concentration dependence. The EC50 values of binding of different antibodies/half antibody molecules to BCMA-expressing positive tumor cells are shown in Table 1 below.

Table 1 EC50 values of binding of antibody/half-antibody molecules to BCMA-expressing positive tumor cells

It can be seen from this that antibody molecules 1 and 3 bind to BCMA-expressing positive tumor cells with lower EC50 values than half antibody molecules 2-1 and 2-2.

Example 4. Binding activity detection of anti-BCMA/anti-P329G bispecific antibody and P329G CAR

This example detects the binding activity of molecule 1 and molecule 3 to Jurkat-NFAT cells expressing P329G CAR.

The different P329G CAR expression vectors prepared in Example 2 were transfected into Lenti-X-293T cells (Takara) at a mass ratio of 3:3:2:2 using the PEI transfection method with structural plasmid pMDLg/pRRE (Addgene, 12251, purchased from Biowind), regulatory plasmid pRSV-rev (Addgene, 12253, purchased from Biowind) and envelope plasmid pMD2G (Addgene, 12259, purchased from Biowind). After 16 hours of transfection, the medium was replaced with fresh DEME medium containing 2% fetal bovine serum (FBS). After continued culture for 48 hours, the cell supernatant was collected, centrifuged to remove cell debris, PEG8000 was added and incubated at 4°C for 16-64 hours to concentrate the lentivirus, and the supernatant was removed after centrifugation again. The lentivirus precipitate was resuspended in T cell culture medium to obtain a lentivirus concentrate, which was packaged and frozen at -80°C. The T cell culture medium is prepared by adding recombinant human interleukin-2 for injection (National Medicine Standard No. S20040020) to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to obtain a T cell culture medium with an IL-2 concentration of 200 IU/ml.

Take an appropriate amount of Jurkat-NFAT cells in the logarithmic growth phase (Jiman Biotechnology (Shanghai) Co., Ltd., GM-C01459), add the lentiviral concentrate obtained by the above method at MOI = 5, and blow the cells evenly, place them in a 37°C _CO2 incubator for static culture, and Jurkat-NFAT cells expressing different P329G CARs can be obtained.

Molecules 1, molecule 3 and control anti-P329G antibody (i.e., anti-P329G IgG, used as control) were prepared into antibody solutions of different concentrations by 5-fold gradient dilution using FACS buffer, and incubated with 1E5 prepared Jurkat-NFAT cells expressing different P329G CARs (CARs expressing extracellular regions of CH2PG, CH2PGAA, CH2PGCH2PG, CH2PGCH3 or CH2PGN297G), respectively, at 4°C for 30 min, and washed once with FACS buffer; the cells were then resuspended with FACS buffer, and Biotin-Protein L (Acro Biosystems, RPL-P814R) was added, and incubated at 4°C for 30 min; after washing twice with FACS buffer, APC-Streptavidin was added, and staining was performed at 4°C for 30-45 min; the cells were washed twice and resuspended with FACS buffer, and detected by flow cytometry, with the antibody concentration as the X-axis and the proportion of APC-positive cell population as the Y-axis to plot and calculate the _EC50 of antibody binding.

The results show that molecule 1 and molecule 3 are bound to CH2PG CAR, CH2PGAA CAR, CH2PGCH2PG CAR, CH2PGCH3CAR or CH2PGN297G CAR. Figure 6 illustrates the binding ability of different concentrations of molecule 1 and molecule 3 to Jurkat-NFAT cells expressing CH2 PG CAR (also known as CH2 P329G CAR).

As shown in FIG6 , both molecule 1 and molecule 3 can recognize and bind to the CH2P329G mutation expressed on the surface of Jurkat-NFAT cells, and the concentration of molecule 1 and molecule 3 is dependent.

The EC50 values of binding of different anti-BCMA/anti-P329G bispecific antibodies to Jurkat-NFAT cells expressing CH2 P329G CAR are shown in Table 2 below.

Table 2 EC50 values of binding of anti-BCMA/anti-P329G bispecific antibodies to Jurkat-NFAT cells expressing CH2 P329G CAR

Example 5. Detection of activation of Jurkat-NFAT cells expressing CH2 P329G CAR by anti-BCMA/anti-P329G bispecific antibodies in vitro

An appropriate amount of CH2 P329G CAR-Jurkat-NFAT cells in the logarithmic growth phase prepared in Example 4 were taken, and the cells were mixed with H929 tumor target cells at an E:T ratio of 3:1, and 10-fold gradient dilutions of different concentrations of molecule 1, molecule 2-1, molecule 2-2, molecule 2-1+molecule 2-2, and molecule 3 were added to a total volume of 75 μL, and cultured at 37°C for about 20 hours. The antibody-mediated target cell-dependent reporter cell activation effect was detected using a luciferase assay kit (Promega, E2620), and the antibody/half-antibody concentration was used as the X-axis and the fluorescence reading change was used as the Y-axis for plotting and analysis.

Figure 7 shows that molecule 1, molecule 2-1 + molecule 2-2, and molecule 3 can all specifically mediate the activation effect of the CH2 P329G CAR reporter cell line, and are antibody concentration-dependent, with no significant difference between molecule 1 and molecule 3. When the two half antibodies of molecule 2, namely molecule 2-1 and molecule 2-2, are used in combination, activation of the Jurkat reporter cell line expressing CH2 P329G CAR can be observed, while the use of the two half antibodies alone cannot induce activation of CH2 P329G CAR-Jurkat reporter cells.

The EC50 values of the specific mediated activation effects of different molecules on the CH2 P329G CAR reporter cell line are shown in Table 3. As can be seen from Table 3, compared with molecules 1 and 3, the EC50 of the combined molecule 2 is about 10 times that of the molecule 1 and 3.

Table 3 EC50 values of the activation effect of anti-BCMA/anti-P329G bispecific antibodies on Jurkat-NFAT cells expressing CH2 P329G CAR

Example 6. In vitro activation detection of anti-BCMA/anti-P329G bispecific antibodies on cells expressing different P329G CAR-Jurkat-NFAT

Jurkat-NFAT cells expressing different P329G CARs were prepared according to the experimental procedures described in Example 4.

An appropriate amount of P329G CAR-Jurkat-NFAT cells with different P329G in the logarithmic growth phase were taken, and the cells were mixed with H929 tumor target cells at an E:T ratio of 3:1, and 10-fold gradient dilutions of different concentrations of molecule 1 and molecule 3 were added to a total volume of 75 μL, and cultured at 37°C for about 6 hours. The antibody-mediated target cell-dependent reporter cell activation effect was detected using a luciferase assay kit (Promega, E2620), and the antibody concentration was used as the X-axis and the fluorescence reading change was used as the Y-axis for plotting and analysis.

Figures 8A and 8B respectively show the activation effects of molecule 1 and molecule 3 on Jurkat reporter cell lines expressing different P329G CARs. The results show that both molecule 1 and molecule 3 can specifically mediate the activation of reporter cell lines containing P329G mutations, including Jurkat reporter cell lines expressing CH2 CAR, CH2CH2 CAR, CH2AA CAR, CH2CH3 CAR, and CH2N297G CAR, and are antibody concentration-dependent, but have no activation effect on Jurkat-NFAT cells expressing CH2ori CAR (CH2 without P329G mutation).

Example 7. Preparation of different P329G CAR-T cells and detection of CAR expression

Recombinant human interleukin-2 for injection (National Medicine Standard No. S20040020) was added to TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) to prepare a T cell culture medium with an IL-2 concentration of 200 IU/ml.

Donor PBMC cells were obtained from ORiCELLS. The specific information is shown in Table 4 below:

Table 4. Related information of donor PBMC cells

PBMC cell number Catalog Number Batch number Donor ID number Donor 10PBMC FPB005F-C PCH20201100006 Z0053

On day 0, the resuscitated PBMCs were sorted using Pan T Cell Isolation Kit (human) (Miltenyi, 130-096-535) to obtain T cells, which were resuspended to a certain density using T cell culture medium and activated by adding TransAct (Miltenyi, 130-111-160).

On the first day, a certain amount of T cells were separated and cultured without adding lentiviral concentrate. This part of the cells was untransduced cells (UNT, un-transduced T cells), and the remaining T cells were added with lentiviral concentrate obtained as described in Example 4 at MOI = 1 to 5 (the lentivirus was a lentivirus encoding CH2ori (SEQ ID NO: 10), CH2PG (SEQ ID NO: 11), CH2PGCH2PG (SEQ ID NO: 13) and CH2PGCH3 (SEQ ID NO: 14)), and the T cells were blown evenly; on the second day, the virus supernatant was removed by centrifugation and the cells were resuspended in fresh T cell culture medium. On the third day, all cells were transferred to G-Rex (WILSONWOLF, catalog number 80240), and an appropriate amount of fresh T cell culture medium was added, and the cells were placed in a 37°C _CO2 incubator for static culture; every 2 to 3 days, the cells were replaced with half of the culture medium with fresh culture medium or IL-2 was directly added, wherein the IL-2 concentration added to the cell culture medium was 200 IU/ml. When the number of cells expanded to about 20-80 times, the cells were harvested after meeting the requirements (generally reaching 2 to ^8x108 cells). After centrifugation to remove the culture medium, the CAR-T cells were used CS10 (Stemcell, 07930) was resuspended and aliquoted, and then programmed to cool to -80°C for cryopreservation.

An appropriate amount of CAR-T cells was taken, washed once with FACS buffer (PBS + 2% FBS), resuspended and added with FACS buffer containing LIVE / DEADFixable Dead Cell Stain, stained at room temperature for 10-15 minutes, washed twice, and PerCP-Cy5.5-CD3, BV605-CD4, PacB-CD8, Alexa Fluor 647-labeled anti-P329G IgG (prepared as in Example 1, for detection of CAR containing P329G mutation) or APC-F (ab ') ₂ Fragment goat anti-human IgG (Jackson ImmunoResearch, 109-136-098; for CH2ori CAR detection) antibody combination was added, stained at 4 ° C for 30-45 minutes, washed twice and resuspended with FACS buffer, and detected by flow cytometry.

Figure 9 shows the expression of CAR in CD3 ⁺ cells, CD4 ⁺ , and CD8 ⁺ T cell subsets 10 days after T cells were transduced with the four CARs constructed in Example 2. The results show that the transduction efficiency of primary T cells is basically the same between different CAR constructs. The only difference that can be observed is CH2CH3 CAR, which is due to its low viral titer, so a lower MOI is used to transduce T cells (MOI=1.7), while the other constructs use a viral MOI=5. In addition, the ratio of CD4 and CD8 expression of CAR is also basically consistent between different constructs.

Example 8. In vitro expansion of different P329G CAR-T cells

As described in Example 7, T cells transduced with different P329G CAR constructs were prepared, and CAR-T cells were cultured in T cell culture medium, and some cell samples were taken out at different time periods, and counted by cell counter to obtain the density, diameter and survival rate parameters of cell growth, and then the total number of cells and expansion multiples were calculated according to the cell culture volume. The expansion of CAR-T cells expressing different P329G was monitored within 10 days.

FIG. 10A shows the changes in the expansion fold of CAR-T cells, FIG. 10B shows the changes in the diameter of CAR-T cells, and FIG. 10C shows the changes in the survival rate of CAR-T cells.

The above results all show that CAR-T cells expressing CH2ori domain, CH2PG domain, CH2PGCH2PG domain, and CH2PGCH3 domain all exhibited relatively consistent cell expansion performance with no significant differences.

Example 9: Detection of dynamic killing effect of CH2 P329G CAR-T cells in vitro

The xCELLigence RTCA MP instrument was used to dynamically detect the killing of target cells by CAR-T cells in real time.

Add 50 μL of culture medium to the E-Plates plate, and after the instrument reads the baseline value, add 50 μL of HT1080 tumor target cells (10,000 cells) expressing BCMA, and place them in the instrument for dynamic detection. At the same time, the CAR-T cells with CH2 P329G extracellular domain prepared in Example 7 (also referred to as CH2 P329G CAR-T cells) were revived and cultured stably at 37°C overnight. After about 24 hours, the CAR-T cells were added to the wells of the corresponding target cells according to E:T of 5:1, and 5-fold gradient dilutions of different concentrations of molecule 1 and molecule 3 solutions were added to a total volume of 200 μL. The xCELLigence RTCA MP instrument system dynamically monitors the killing of target cells by CH2P329G CAR-T cells for 40-96 hours.

Figure 11A (molecule 1) and Figure 11B (molecule 3) respectively show the dynamic killing effect of CH2 P329G CAR-T on tumor cells mediated by molecule 1 and molecule 3 in the presence of target cells.

According to the results, it can be found that molecules 1 and 3 can mediate the inhibitory effect of CH2P329GCAR-T cells on tumor cell growth at a lower antibody concentration (1.6nM), and at a concentration higher than this, they can induce CH2P329GCAR-T cells to completely lyse tumor target cells.

Example 10. Detection of dynamic killing effects of different P329G CAR-T cells in vitro

The xCELLigence RTCA MP instrument was used to dynamically and in real time detect the killing of target cells by CAR-T cells. 50 μL of culture medium was added to the E-Plates plate. After the instrument read the baseline value, 50 μL of HT1080 tumor target cells (10,000 cells) expressing BCMA were added and placed in the instrument for dynamic detection. At the same time, the four different CAR-T cells prepared in Example 7 were resuscitated and cultured stably at 37°C overnight. After about 24 hours, the positive rates of all CAR-T cells were adjusted to be consistent using UNT cells, and four different CAR-T cells were added to the wells of the corresponding target cells according to E:T of 5:1, and the corresponding concentrations of molecule 1, half antibody molecule 2-1 + half antibody molecule 2-2, and molecule 3 solutions were added to a total volume of 200 μL, and the xCELLigence RTCA MP instrument system dynamically monitored the killing of target cells by different CAR-T cells for 40-96 hours.

Figure 12A (molecule 1), Figure 12B (molecule 3) and Figure 12C (molecule 2 formed by half antibody molecule 2-1 + half antibody molecule 2-2) respectively show the dynamic killing effects of different CAR-T cells on tumor cells mediated by molecule 1, molecule 3 and molecule 2 in the presence of target cells. According to the results, it can be found that molecules 1 and molecule 3 can specifically mediate the complete lysis of tumor target cells by P329G CAR-T cells with extracellular domains of CH2, CH2CH2 and CH2CH3 domains under 8nM conditions, but have no induction activation effect on CAR-T cells expressing CH2ori (CH2 without P329G mutation).

At the same time, it can be observed that when using the two half antibodies in molecule 2, that is, using molecule 2-1 and molecule 2-2, under 200nM conditions, both molecule 2-1 and molecule 2-2 can bind to the BCMA antigen on the target cells, and can form functional anti-P329G, and then bind to P329G CAR-T cells expressing extracellular domains of CH2, CH2CH2 and CH2CH3, and induce CAR-T cells to exert a killing effect on tumor target cells, especially inducing P329G CAR-T cells with extracellular domains of CH2CH2 and CH2CH3 to completely lyse tumor target cells.

Sequence Listing

Claims (21)

1. An anti-BCMA/anti-P329G bispecific antibody, the anti-BCMA/anti-P329G bispecific antibody comprising two heavy chains and two light chains, and i) F(ab) ₂ with specific binding to BCMA and a heavy chain variable region located at the C-terminus of the CH3 domain of one heavy chain that specifically binds to the P329G mutated anti-P329G heavy chain variable region located at the CH3 domain of the other heavy chain The C-terminal anti-P329G light chain variable region specifically binds to the P329G mutation; or ii) Having two half-antibodies consisting of one heavy chain and one light chain, one of which has an N-terminal Fab that specifically binds BCMA and a specific Fab located between the C-terminal of the heavy chain Fab and the N-terminal of the CH2 domain The other half-antibody has an N-terminal Fab that specifically binds BCMA and a P329G mutation located between the C-terminus of the heavy chain Fab and the N-terminus of the CH2 domain. The light chain variable region of an anti-P329G; or iii) An anti-P329G scFv having F(ab) ₂ that specifically binds BCMA and a mutation that specifically binds P329G, in which the C-terminus of the CH3 domain of one heavy chain is connected to the N-terminus of the anti-P329G scFv. 2. The anti-BCMA/anti-P329G bispecific antibody according to claim 1, wherein a) The Fab or F(ab) ₂ that specifically binds BCMA comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence SSSYYWT according to Kabat numbering (SEQ ID NO: 25 ), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR H2 with the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26), or the CDR H2 A variant with no more than 2 amino acid changes or no more than 1 amino acid change; and a CDR H3 shown in the amino acid sequence DRGDQILDV (SEQ ID NO: 27), or a CDR H3 with no more than 2 amino acid changes or no more than A variant of 1 amino acid change; the light chain variable region includes CDR L1 shown according to the Kabat numbered amino acid sequence RASQSISRYLN (SEQ ID NO: 28), or no more than 2 amino acid changes of the CDR L1 or no Variants with more than 1 amino acid change; CDR L2 represented by the amino acid sequence AASSLQS (SEQ ID NO: 29), or variants of the CDR L2 with no more than 2 amino acid changes or no more than 1 amino acid change; and amino acids The CDR L3 shown in the sequence QQKYFDIT (SEQ ID NO: 30), or a variant of the CDR L3 with no more than 2 amino acid changes or no more than 1 amino acid change; wherein said amino acid change is the addition, deletion or substitution of amino acids; For example, the heavy chain variable region includes CDR H1 represented by the amino acid sequence SSSYYWT (SEQ ID NO: 25) according to Kabat numbering; CDR H2 represented by the amino acid sequence SISIAGSTYYNPSLKS (SEQ ID NO: 26); and the amino acid sequence DRGDQILDV CDR H3 shown in (SEQ ID NO: 27); the light chain variable region includes CDR L1 shown according to the amino acid sequence RASQSISRYLN (SEQ ID NO: 28) numbered by Kabat; the amino acid sequence AASSLQS (SEQ ID NO: 29 CDR L2 represented by ); and CDR L3 represented by the amino acid sequence QQKYFDIT (SEQ ID NO: 30); For example, the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 23 Sequence identity, and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequences with 99% identity; For example, the heavy chain variable region is the sequence of SEQ ID NO: 23, and the light chain variable region is the sequence of SEQ ID NO: 24; b) The anti-P329G or anti-P329G scFv that specifically binds to the P329G mutation comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence RYWMN according to Kabat numbering (SEQ ID NO: 31 ), or a variant of the CDR H1 with no more than 2 amino acid changes or no more than 1 amino acid change; CDR H2 with the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 32) , or a variant of the CDR H2 with no more than 2 amino acid changes or no more than 1 amino acid change; and a CDR H3 represented by the amino acid sequence PYDYGAWFAS (SEQ ID NO: 33), or a CDR H3 with no more than 2 changes A variant with an amino acid change or no more than 1 amino acid change; the light chain variable region includes a light chain complementarity determining region (CDR L) 1, Or a variant of the CDR L1 with no more than 2 amino acid changes or no more than 1 amino acid change; the CDR L2 shown in the amino acid sequence GTNKRAP (SEQ ID NO: 35), or the CDR L2 with no more than 2 amino acids Variants that change or have no more than 1 amino acid change; and CDRL3 represented by the amino acid sequence ALWYSNHWV (SEQ ID NO: 36), or variants of the CDR L3 that have no more than 2 amino acid changes or no more than 1 amino acid change. ; wherein said amino acid change is the addition, deletion or substitution of amino acids; For example, the heavy chain variable region includes CDR H1 represented by the amino acid sequence RYWMN (SEQ ID NO: 31) according to Kabat numbering; CDR H2 represented by the amino acid sequence EITPDSSTINYAPSLKG (SEQ ID NO: 32); and the amino acid sequence PYDYGAWFAS CDR H3 shown in (SEQ ID NO: 33); the light chain variable region includes CDR L1 shown in the amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 34) according to Kabat numbering; amino acid sequence GTNKRAP (SEQ ID NO: 35 CDR L2 represented by ); and CDR L3 represented by the amino acid sequence ALWYSNHWV (SEQ ID NO: 36); For example, the heavy chain variable region comprises or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 21 Sequence identity, and the light chain variable region comprises or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or Sequences with 99% identity; For example, the heavy chain variable region is the sequence of SEQ ID NO:21, and the light chain variable region is the sequence of SEQ ID NO:22. 3. The anti-BCMA/anti-P329G bispecific antibody according to claim 1 or 2, the heavy chain of the anti-BCMA/anti-P329G bispecific antibody is IgG1 or IgG4 class; more preferably, it is IgG1 class heavy chain. Chain; wherein the two heavy chains respectively comprise bulges or holes in their respective Fc domains, and the bulges or holes in the Fc domain of one heavy chain can be respectively placed in the Fc structure of another heavy chain in the cavities or bulges in the domain, whereby the heavy chains of the anti-BCMA/anti-P329G bispecific antibody form a "knotted" stable association with each other, e.g., between the two heavy chains The amino acid substitution T366W is included in one heavy chain and the amino acid substitutions T366S, L368A and Y407V (EU numbering) are included in the other of the two heavy chains. 4. The anti-BCMA/anti-P329G bispecific antibody according to claim 1, comprising: i) Two light chains as shown in SEQ ID NO: 2 and two heavy chains as shown in SEQ ID NO: 1 and SEQ ID NO: 3 respectively, or at least 80% or 85% with any of the sequences. , 90%, 92%, 95%, 97%, 98%, 99% or more identical sequences; or ii) Two light chains as shown in SEQ ID NO: 2 and two heavy chains as shown in SEQ ID NO: 4 and SEQ ID NO: 5 respectively, or at least 80%, 85% with any of the sequences , 90%, 92%, 95%, 97%, 98%, 99% or more identical sequences; or iii) Two light chains as shown in SEQ ID NO: 2 and two heavy chains as shown in SEQ ID NO: 6 and SEQ ID NO: 7 respectively, or at least 80% or 85% with any of the sequences. , 90%, 92%, 95%, 97%, 98%, 99% or higher identical sequences. 5. An isolated nucleic acid encoding the anti-BCMA/anti-P329G bispecific antibody of any one of claims 1-4. 6. A vector comprising the nucleic acid of claim 5, preferably said vector is an expression vector. 7. A host cell comprising the nucleic acid of claim 5 or the vector of claim 6, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from the group consisting of E. coli cells, yeast cells, mammalian cells or suitable As for other cells for preparing antibodies, most preferably, the host cell is HEK293 cells or CHO cells. 8. A method for preparing the anti-BCMA/anti-P329G bispecific antibody of any one of claims 1-4, said method comprising encoding the anti-BCMA of any one of claims 1-4. /anti-P329G bispecific antibody nucleic acid, culturing host cells introduced with an expression vector encoding the nucleic acid encoding the anti-BCMA/anti-P329G bispecific antibody according to any one of claims 1 to 4, and isolating the an anti-BCMA/anti-P329G bispecific antibody, optionally the method further comprising recovering the anti-BCMA/anti-P329G bispecific antibody from the host cell; or The method includes culturing and introducing the nucleic acid encoding the two half-antibodies in the anti-BCMA/anti-P329G bispecific antibody of any one of claims 1-4 under conditions suitable for expression. The host cell of the nucleic acid expression vector of one of the two half-antibodies of the anti-BCMA/anti-P329G bispecific antibody described in any one of 4, and the host cell of the nucleic acid expression vector of the anti-BCMA/anti-P329G bispecific antibody. A half-antibody, and a host cultured with an expression vector introduced with an expression vector encoding the other half-antibody of the two half-antibodies of the anti-BCMA/anti-P329G bispecific antibody according to any one of claims 1-4 cells, isolating another half-antibody of said anti-BCMA/anti-P329G bispecific antibody, optionally the method further comprising recovering said two half-antibodies from said host cell; a combination of said two half-antibodies Constitute anti-BCMA/anti-P329G bispecific antibody. 9. A chimeric antigen receptor (CAR) polypeptide that binds anti-P329G, the chimeric antigen receptor polypeptide comprising: (i) an extracellular domain, which is a mutant Fc domain or a fragment thereof or a combination of fragments thereof, wherein the amino acid at position P329 according to EU numbering is mutated to glycine (G); For example, the mutant Fc domain is a mutant Fc domain of an IgG1, IgG2, IgG3 or IgG4 antibody. Preferably, the mutant Fc domain is a mutant Fc domain of an IgG1 or IgG4 antibody; more preferably, the mutation The Fc domain is the mutated Fc domain of IgG1 antibody; For example, the mutant Fc domain fragment or combination of fragments thereof is a single CH2 domain comprising the P329G mutation; A single CH2 domain containing the P329G mutation and a partially truncated N-terminus; CH2CH2 domain consisting of two CH2 domains containing the P329G mutation connected together; A CH2CH3 domain consisting of a CH2 domain containing the P329G mutation connected to a normal CH3 domain; or A CH2CH3 domain consisting of a CH2 domain containing P329G and N297G mutations connected to a normal CH3 domain; (ii) CD8 transmembrane domain or a variant thereof with 1-10 amino acid modifications; (iii) 4-1BB costimulatory domain or a variant thereof with 1-10 amino acid modifications; and (iv) CD3ζ activation domain or a variant thereof with 1-10 amino acid modifications. 10. The chimeric antigen receptor polypeptide according to claim 9, said chimeric antigen receptor further comprising a G4S linker (SEQ ID NO: 17) between said (i) and said (ii); And/or the signal peptide sequence located at the N-terminus of (i), for example, the signal peptide sequence shown in SEQ ID NO: 16. 11. The chimeric antigen receptor polypeptide according to claim 9 or 10, comprising: (i) As set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 or at least 90%, 91%, 92%, 93% thereof , a P329G mutant Fc domain or a fragment thereof or a combination of fragments thereof that is 94%, 95%, 96%, 97%, 98% or 99% identical; (ii) CD8 transmembrane domain as shown in SEQ ID NO: 18 or a variant thereof having 1-5 amino acid modifications; (iii) 4-1BB costimulatory domain as shown in SEQ ID NO: 19 or a variant thereof having 1-5 amino acid modifications; and (iv) CD3ζ activation domain as shown in SEQ ID NO: 20 or a variant thereof with 1-5 amino acid modifications. 12. A nucleic acid molecule encoding the chimeric antigen receptor polypeptide of any one of claims 9-11. 13. A vector comprising the nucleic acid molecule of claim 12, for example, the vector is selected from the group consisting of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenoviral vectors or retroviral vectors. 14. A cell comprising the CAR polypeptide of any one of claims 9-11, the nucleic acid molecule of claim 12, or the vector of claim 13, which cell is, for example, an immune effector cell, for example, The immune effector cells are T cells and NK cells. For example, the T cells are autologous T cells or allogeneic T cells. For example, the immune effector cells are expressions prepared from T cells and NK cells isolated from human PBMC. T cells and NK cells of the CAR polypeptide according to any one of claims 9-11. 15. A method of making the cell of claim 14, comprising transducing the cell with the vector of claim 13. 16. Pharmaceutical combinations containing (i) Selected from the nucleic acid molecule of claim 12, the vector of claim 13, the cell of claim 14, and any combination thereof; and (ii) the anti-BCMA/anti-P329G bispecific antibody of any one of claims 1-4; and Pharmaceutical excipients are optionally available. 17. Use of the pharmaceutical combination of claim 16 for the preparation of a medicament for the treatment of BCMA-related diseases, such as cancers expressing or overexpressing BCMA. 18. Use of a pharmaceutical combination according to claim 16 for the treatment of a disease associated with BCMA in a subject, preferably the disease associated with BCMA is, for example, a cancer that expresses or overexpresses BCMA. 19. A drug complex consisting of (i) Immune effector cells (eg, T cells, NK cells) expressing the CAR polypeptide of any one of claims 9-11; and (ii) The anti-BCMA/anti-P329G bispecific antibody according to any one of claims 1-4 A complex produced by binding of the P329G mutation contained in the extracellular domain of the CAR polypeptide to the anti-P329G of the anti-BCMA/anti-P329G bispecific antibody; For example, wherein the immune effector cells are T cells or NK cells prepared from autologous T cells, NK cells or allogeneic T cells or NK cells expressing the CAR polypeptide according to any one of claims 9-11, such as , the immune effector cells are T cells or NK cells prepared from T cells or NK cells isolated from human PBMC and expressing the CAR polypeptide according to any one of claims 9-11. 20. Use of a pharmaceutical complex according to claim 19 for the treatment of a disease associated with BCMA in a subject, preferably the disease associated with BCMA is, for example, a cancer that expresses or overexpresses BCMA. 21. Use of the pharmaceutical complex of claim 19 for the preparation of a medicament for the treatment of BCMA-related diseases, such as cancers expressing or overexpressing BCMA.