RU2810092C2 - Chimeric antigene receptor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a chimeric antigen receptor (CAR) that binds B-cell maturation antigen (BCMA), as well as a nucleic acid expression construct and a cell containing a nucleic acid molecule encoding the said CAR. A vector containing the above expression construct is also disclosed. The invention also relates to a method of producing a cytolytic immune cell having the ability to kill a target cell expressing BCMA, as well as a composition containing the above cell.

EFFECT: invention is effective for the treatment of cancer.

14 cl, 13 dwg, 2 tbl, 10 ex

Description

TECHNICAL FIELD

The present invention relates to a chimeric antigen receptor (CAR) that binds to a low density target antigen, such as B cell maturation antigen (BCMA).

BACKGROUND OF THE ART

Multiple myeloma

Multiple myeloma (myeloma) is a malignant tumor of the bone marrow that arises from plasma cells. Populations of abnormal plasma cells accumulate in the bone marrow, where they interfere with the production of normal blood cells. Myeloma is the second most common hematologic malignancy in the United States (after non-Hodgkin's lymphoma), accounting for 13% of hematologic malignancies and 1% of all cancers. The disease is burdensome in terms of patient suffering and medical expenses as it is associated with pathological fractures, susceptibility to infection, renal failure and subsequent bone marrow failure before death.

Unlike many types of lymphoma, myeloma currently has no cure. Standard chemotherapy agents used for lymphoma are generally ineffective in myeloma. In addition, due to loss of CD20 expression on plasma cells, rituximab cannot be used in this disease. Newer agents such as bortezomib and lenolidomide are partially effective but do not provide long-term remission.

Therefore, there is a need for alternative agents for the treatment of myeloma that have higher efficacy and improved long-term effects.

CHIMERIC ANTIGENE RECEPTORS (CAR)

Chimeric antigen receptors are proteins that, in their conventional format, confer monoclonal antibody (Mab) specificity to T cell effector function. Their common form is a type I transmembrane protein with an antigen recognition amino terminus, a spacer, and a transmembrane domain, all connected to a composite endodomain that conveys T cell survival and activation signals.

The most common form of these molecules uses single-chain variable fragments (scFvs) derived from monoclonal antibodies to recognize the target antigen. scFv binds to the signaling endodomain via a spacer and transmembrane domain. Such molecules cause T cell activation in response to scFv recognition of its target. When T cells express such a CAR, they recognize and kill target cells expressing the target antigen. Several CARs against tumor-associated antigens have been developed, and adoptive transfer approaches of such CAR-expressing T cells are currently in clinical trials for the treatment of various types of cancer. Carpenter et al. (2013, Clin Cancer Res 19(8) 2048-60) describe a CAR that contains an anti-B cell maturation antigen (BCMA) scFv.

BCMA is a transmembrane protein that is predominantly expressed by mature lymphocytes, i.e. memory B cells, plasmablasts and bone marrow plasma cells. BCMA is also expressed on multiple myeloma cells.

Carpenter et al. demonstrate that transduced T cells expressing anti-BCMA CAR are able to specifically kill myeloma cells isolated from a patient with plasmacytoma or myeloma.

Despite the successes demonstrated by CAR approaches using antibodies against BCMA, when targeting this antigen, special attention must be paid to the fact that BCMA is present at very low density on myeloma cells compared to, for example, CD19 expression on lymphoma cells. Despite the success demonstrated in the clinic with CAR T cell-mediated therapies targeting common target antigens such as CD19 and GD2, there is evidence that chimeric antigen receptors are unable to generate a signal in response to antigens that have very low density.

For example, results from a study of CAR T cells targeting anaplastic lymphoma kinase (ALK) showed that CAR T cells had limited antitumor efficacy in two xenograft models of human neuroblastoma. It was shown that cytokine production was highly dependent on target density (ALK) and that target density (ALK) on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells (Walker et al. (2017) Mol. Ther. 25, 2189-2201).

In another study, anti-CD22 CAR T cells were used to treat relapsed and/or refractory B-cell precursor lymphoblastic leukemia (B-ALL), although some dose-dependent antileukemic activity was observed, relapses occurred in some cases. Relapses were associated with decreased density of CD22 sites, which was thought to mediate the ability of CD22+ cells to evade killing by CD22 CAR T cells (Fry et al. (2017) Nat. Med. doi:10.1038/nm.4441).

There is a hierarchy in CAR T cell activation from target killing to cytokine release to proliferation. A CAR T cell can kill a target cell that has low antigen density, but may not achieve full activation.

Therefore, there is a need for alternative CAR T cell approaches that can kill target cells expressing low density target antigen.

DESCRIPTION OF FIGURES

Figure 1: BCMA expression in patients with multiple myeloma

Figure 2: Different formats of the chimeric antigen receptor binding domain

(a) Fab CAR format; (b) dAb CAR format; (c) scFv CAR format

Figure 3: Schematic diagram illustrating the interaction of a classical CAR T cell and a target cell expressing a low density target antigen. (a) CAR T cell membrane in normal state; (b) the CAR T cell membrane forms an immune synapse with the cell with a high density of target expression; (c) CAR T cell membrane in response to a target cell that has low antigen density.

Figure 4: Schematic diagram illustrating a constitutively active chimeric antigen receptor. The chimeric transmembrane protein contains a dimerization domain and a cytokine receptor endodomain. The presented embodiment demonstrates a "Fab" type architecture in which the dimerization domain contains heavy and light chain constant regions in the manner of an antibody. Stable dimerization of these two domains brings the common interleukin-2 (IL-2) receptor γ-chain into proximity with the IL-2 receptor β-chain or the IL-7 receptor α-chain, resulting in constitutive cytokine signaling.

Figure 5: Results of a cytotoxicity test comparing CARs against BCMA in scFv and FabCAR formats. CAR-expressing cells were co-cultured with non-BCMA-expressing (SupT1 NT), low-BCMA-expressing (SupT1BCMAlow), or very low-BCMA-expressing (JeKo-1) target cells in a ratio of 1:4 (Figure 5A) or 1: 8 (Figure 5B). bb2121, a previously described anti-BCMA CAR, was used as a positive control. After 24 hours, target cell killing was assessed by flow cytometry (FACS), and the number of target cells was normalized to the number of non-transfected T cells (NT).

Figure 6: Results of a cytotoxicity test comparing CARs against BCMA in scFv and FabCAR formats. CAR-expressing cells were cocultured with target cells not expressing BCMA (SupT1 NT), low BCMA expressing (SupT1BCMAlow), or very low BCMA expressing (JeKo-1) in a ratio of 1:4 (Figure 6A) or 1: 8 (Figure 6B). bb2121, a previously described anti-BCMA CAR, was used as a positive control. After 72 hours, target cell killing was assessed by FACS, and the number of target cells was normalized to the number of non-transfected T cells (NT).

Figure 7: Interferon-γ (IFN-γ) production by T cells expressing anti-BCMA CAR in either scFv or FabCAR formats after co-culture with target cells.

Cells expressing CAR were co-cultured with target cells not expressing BCMA (Figure 7A) or with low expression of BCMA (Figure 7B) at a ratio of 1:4 or 1:8. bb2121, a previously described anti-BCMA CAR, was used as a positive control. After 24 hours, IFN-γ production was assessed by enzyme-linked immunosorbent assay (ELISA)

Figure 8: Number of CD4 and CD8 T cells expressing anti-BCMA CAR in scFv or FabCAR format after co-culture with target cells.

CAR-expressing cells were co-cultured with non-BCMA-expressing (SupT1 NT), low-BCMA-expressing (SupT1BCMAlow), or very low-BCMA-expressing (JeKo-1) target cells, or MM.1s target cells at a ratio of 1 :1 within 96 hours. The number of whole CD4 (Figure 8A) and CD8 (Figure 8B) T cells was assessed by gating for CD3 and the RQR8 marker, then CD8+ or CD8−.

Figure 9: Schematic diagram illustrating a cleavable FabCAR

The cleavable FabCAR contains two antigen-binding domains: outer (membrane-distal) binding domain 1 and inner (membrane-proximal) binding domain 2. Binding domains 1 and 2 are connected by a cleavable linker. Cleavage of the linker, for example by matrix metalloproteinase (MMP), eliminates binding domain 1. Without cleavage, with FabCAR intact, the internal binding domain is partially covered by the presence of the external domain due to steric hindrance. The internal domain is activated after cleavage of the heavy and light chains.

Figure 10: Schematic representation of the soluble antibody described in Example 7

The soluble antibody was designed with two variable domains and an external anti-BCMA binding domain (anti-BCMA D8) and an internal anti-CD19 binding domain (anti-CD19 CAT). The MMP-9 cleavable linker was inserted between the VH domains on one strand and the VL domains on the other strand. The VH-containing chain also contained the Fc portion of the antibody.

Figure 11: The graph shows the percentage degradation of soluble antibody (D8-MMP9-CAT19) over time after incubation in the presence of MMP-9. An equivalent antibody with a randomized linker that does not contain the MMP-9 cleavage site (D8-randomized-CAT19) was used as a negative control.

Figure 12: Graph shows ELISA results of CD19 binding with soluble antibody (D8-MMP9-CAT19) or negative control with randomized linker (D8-randomized-CAT19) without and after exposure to MMP-9.

Figure 13: The graph shows the kinetics of CD19 binding by soluble antibody without exposure (D8-MMP9-CAT19) and after exposure to MMP-9 (D8-MMP9-CAT19 cleaved). Anti-CD-19 monoclonal antibody (CAT19 IgG) was used as a positive control.

BRIEF DESCRIPTION OF ASPECTS OF THE INVENTION

In the present invention, we have found that when targeting CAR T cells to a target antigen having a low density, the use of a CAR with an antigen binding Fab domain allows for more efficient CAR-mediated signaling.

Thus, the first aspect of the present invention provides a chimeric antigen receptor (CAR) that binds a target antigen having a low density, wherein the CAR comprises a Fab antigen binding domain.

The target antigen may be expressed at an average density of less than 1500 copies per target cell.

The target antigen can be selected from: ROR1, Tyrp-1, TACI, ALK and BCMA. For example, the target antigen may be BCMA.

In an alternative embodiment of the first aspect of the present invention, the antigen binding domain of a Fab may comprise a first binding domain that binds to a first target antigen and a second binding domain that binds to a second target antigen.

The first target antigen or the second target antigen may be BCMA.

In the present embodiment, the FabCAR may comprise a cleavable linker between the first and second binding domains.

The linker can be cleaved by an agent such as an enzyme. For example, the linker can be cleaved by matrix metalloproteinase (MMP). The agent can be administered to a subject before or after administration of the CAR expressing cells of the present invention to cleave the first binding domain and release the second binding domain. Alternatively, cleavage may occur naturally in a specific region of the body if a cleavage agent (eg, an enzyme) is expressed in that region. This area may be, for example, an area of inflammation or an area of tumor.

A second aspect of the present invention provides a nucleic acid sequence that encodes a CAR according to the first aspect of the present invention.

In a third aspect of the present invention, there is provided a nucleic acid construct that encodes a CAR according to the first aspect of the present invention and has one of the following general structures:

VH-CH-spacer-TM-endo-coexpr-VL-CL;

VL-CL-coexpr-VH-CH-spacer-TM-endo;

VL-CL-spacer-TM-endo-coexpr-VH-CH; or

VH-CH-coexpr-VL-CL-spacer-TM-endo;

Where:

VH is a nucleic acid sequence encoding a heavy chain variable region;

CH is a nucleic acid sequence encoding a heavy chain constant region;

spacer is a nucleic acid encoding a spacer;

TM is a nucleic acid sequence encoding a transmembrane domain;

endo is a nucleic acid sequence encoding an endodomain;

coexpr is a nucleic acid sequence allowing co-expression of the first and second polypeptides;

VL is a nucleic acid sequence encoding a light chain variable region; And

CL is a nucleic acid sequence encoding a light chain constant region.

In an alternative embodiment of the third aspect of the present invention, the nucleic acid construct may have one of the following general structures:

VH1-L1-VH2-CH-spacer-TM-endo-coexpr-VL1-L2-VL2-CL;

VL1-L1-VL2-CL-coexpr-VH1-L2-VH2-CH-spacer-TM-endo;

VL1-L1-VL2-CL-spacer-TM-endo-coexpr-VH1-L2-VH2-CH; or

VH1-L1-VH2-CH-coexpr-VL1-L2-VL2-CL-spacer-TM-endo;

Where:

VH1 is a nucleic acid sequence encoding the heavy chain variable region of the first binding domain;

L1 and L2, which may be the same or different, are cleavable linkers;

VH2 is a nucleic acid sequence encoding the heavy chain variable region of the second binding domain;

CH is a nucleic acid sequence encoding a heavy chain constant region;

spacer is a nucleic acid encoding a spacer;

TM is a nucleic acid sequence encoding a transmembrane domain;

endo is a nucleic acid sequence encoding an endodomain;

coexpr is a nucleic acid sequence allowing co-expression of the first and second polypeptides;

VL1 is a nucleic acid sequence encoding the light chain variable region of the first binding domain;

VL2 is a nucleic acid sequence encoding the light chain variable region of the second binding domain; And

CL is a nucleic acid sequence encoding a light chain constant region.

The nucleic acid construct may also encode a second chimeric antigen receptor with an antigen binding domain that is a domain antibody (dAb), scFv, or Fab.

The second chimeric antigen receptor can bind to one of the following antigens: CD19, FcRL5 and TACI.

In a fourth aspect of the present invention, there is provided a vector that contains a nucleic acid sequence according to the second aspect of the present invention or a nucleic acid construct according to the third aspect of the present invention.

In a fifth aspect of the present invention, there is provided a cell that expresses a CAR according to the first aspect of the present invention.

The cell may express a CAR according to the first aspect of the present invention and a second chimeric antigen receptor as defined above.

The cell may also express a constitutively active cytokine receptor.

A sixth aspect of the present invention provides a method for producing a cell according to the fifth aspect of the present invention, which includes the step of introducing a nucleic acid sequence according to the second aspect of the present invention; a nucleic acid construct according to the third aspect of the present invention or a vector according to the fourth aspect of the present invention into a cell ex vivo.

A seventh aspect of the present invention provides a pharmaceutical composition that contains a plurality of cells according to the fifth aspect of the present invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

In an eighth aspect of the present invention, there is provided a method for treating cancer, which includes the step of administering to a subject a pharmaceutical composition according to the seventh aspect of the present invention.

The cancer may be multiple myeloma.

A ninth aspect of the present invention provides a pharmaceutical composition according to the seventh aspect of the present invention for use in the treatment of cancer.

A tenth aspect of the present invention provides the use of a cell according to the fifth aspect of the present invention in the production of a pharmaceutical composition for the treatment of cancer.

The present invention provides chimeric antigen receptors that exhibit improved CAR-mediated signaling and more efficient killing of target cells when targeting antigens expressed at low density on the target cell. Such antigens are difficult to target with classical CARs as they form suboptimal T cell:target cell synapses (Figure 3).

The ability to target such antigens opens up entirely new possibilities in cancer treatment. Many potentially useful cancer target antigens, such as ROR1, Tyrp-1, TACI, ALK, and BCMA, have low expression densities on target cells. The present invention provides improved designs for targeting these antigens that allow them to be used as individual targets and, importantly, included in multi-antigen targeting strategies to improve the efficacy and safety of CAR T cell applications.

ADDITIONAL ASPECTS

The present invention also provides aspects that are summarized in the numbered paragraphs below.

1. A chimeric antigen receptor (CAR) that contains an antigen binding domain Fab comprising a first binding domain that binds to a first target antigen and a second binding domain that binds to a second target antigen.

2. A CAR according to paragraph 1, which contains a cleavable linker between the first and second binding domains.

3. CAR according to paragraph 2, characterized in that the linker can be cleaved by matrix metalloproteinase (MMP).

4. A CAR as defined in any of the preceding paragraphs, wherein the first target antigen is expressed in a tumor region such that when a CAR-expressing cell is administered to a patient having cancer, the cell homing occurs to the tumor region in the subject's body.

5. A CAR according to any of the preceding paragraphs, characterized in that the second target antigen is expressed in the tumor region as well as in one or more normal tissues.

6. CAR according to any of the previous paragraphs, characterized in that the first and/or second target antigen is selected from the following group: BCMA, ROR1, Tyrp-1, TACI, ALK, ErbB2, MUC1, CD33, CD123, PSMA, EpCAM, GD2, NCAM, folate binding protein and MUC16.

7. CAR according to any of paragraphs 1-5, characterized in that the first target antigen/second target antigen is selected from one of the following antigen pairs: ErbB2 and MUC1; CD33 and CD123; PSMA and EpCAM; GD2 and NCAM; folate binding protein and MUC16.

8. CAR according to paragraph 7, characterized in that the first target antigen/second target antigen is one of the pairs of antigens presented in Table 2.

9. A nucleic acid sequence that encodes a CAR according to any of the previous paragraphs.

10. A nucleic acid construct that encodes a CAR according to any of paragraphs 1-8 and has one of the following general structures:

VH1-L1-VH2-CH-spacer-TM-endo-coexpr-VL1-L2-VL2-CL;

VL1-L1-VL2-CL-coexpr-VH1-L2-VH2-CH-spacer-TM-endo;

VL1-L1-VL2-CL-spacer-TM-endo-coexpr-VH1-L2-VH2-CH; or

VH1-L1-VH2-CH-coexpr-VL1-L2-VL2-CL-spacer-TM-endo,

Where:

VH1 is a nucleic acid sequence encoding the heavy chain variable region of the first binding domain;

L1 and L2, which may be the same or different, are cleavable linkers;

VH2 is a nucleic acid sequence encoding the heavy chain variable region of the second binding domain;

CH is a nucleic acid sequence encoding a heavy chain constant region;

spacer is a nucleic acid encoding a spacer;

TM is a nucleic acid sequence encoding a transmembrane domain;

endo is a nucleic acid sequence encoding an endodomain;

coexpr is a nucleic acid sequence allowing co-expression of the first and second polypeptides;

VL1 is a nucleic acid sequence encoding the light chain variable region of the first binding domain;

VL2 is a nucleic acid sequence encoding the light chain variable region of the second binding domain; And

CL is a nucleic acid sequence encoding a light chain constant region.

11. A vector that contains a nucleic acid sequence according to paragraph 9 or a nucleic acid construct according to paragraph 10.

12. A cell that expresses a CAR according to any of paragraphs 1-8.

13. The method of obtaining a cell according to paragraph 12, which includes the step of introducing a nucleic acid sequence according to paragraph 9; nucleic acid constructs according to paragraph 10 or vectors according to paragraph 11 into a cell ex vivo.

14. A pharmaceutical composition which contains a plurality of cells according to paragraph 12 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

15. A method of treating cancer, which includes the step of administering to a subject a pharmaceutical composition according to paragraph 14.

16. Pharmaceutical composition according to paragraph 14 for use in the treatment of cancer.

17. Use of a cell according to paragraph 12 in the production of a pharmaceutical composition for the treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

CHIMERIC ANTIGENE RECEPTORS

The present invention relates to a chimeric antigen receptor with a Fab antigen binding domain.

The classical chimeric antigen receptor (CAR) is a chimeric type I transmembrane protein that connects an extracellular antigen recognition domain (binding element) to an intracellular signaling domain (endodomain). The binding element is typically a single chain variable fragment (scFv) derived from a monoclonal antibody (Mab), but may be based on other formats containing an antibody-like antigen-binding site. The spacer domain is usually required to isolate the binding element from the membrane to ensure its proper orientation. The Fc fragment of immunoglobulin G1 (IgG1) is usually used as a spacer domain. Depending on the antigen, more compact spacers may be suitable, eg the CD8α stem and even the isolated IgG1 hinge region alone. The transmembrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

In early CAR designs, endodomains were derived from intracellular γ-chain fragments of FcεR1 or CD3ζ. Consequently, these first-generation receptors conveyed an immunological signal 1 that was sufficient to trigger the killing of cognate target cells by T cells, but did not provide the full activation of T cells necessary for their proliferation and survival. To overcome this limitation, composite endodomains were developed: the fusion of the intracellular part of the T cell costimulatory molecule with the same fragment of CD3ζ formed the basis of second-generation receptors, which, after antigen recognition, can simultaneously transmit activating and costimulatory signals. The CD28 domain is most often used as a co-stimulatory domain. This provides the most potent costimulatory signal, namely immunological signal 2, which triggers T cell proliferation. Several receptors that contain endodomains of the tumor necrosis factor (TNF) receptor family have also been described, such as the closely related OX40 and 41BB, which transmit survival signals. To date, even more powerful third-generation CARs have been described, the endodomains of which are capable of transmitting activation, proliferation and survival signals.

When a CAR binds to a target antigen, it results in the transmission of an activating signal to the T cell on which it is expressed. In this way, CAR ensures the specificity and cytotoxicity of the T cell against tumor cells expressing the target antigen.

Therefore, CARs typically contain: (i) an antigen binding domain; (ii) spacer; (iii) a transmembrane domain; and (iii) an intracellular domain that contains or is associated with a signaling domain.

A CAR may have a general structure:

Antigen-binding domain - spacer - transmembrane domain - intracellular signaling domain (endodomain).

Antigen binding domain

The antigen-binding domain is the part of the chimeric receptor that recognizes antigen. In a classic CAR, the antigen binding domain contains: a single chain variable fragment (scFv) derived from a monoclonal antibody (see Figure 2c). CARs with domain antibody (dAb) or VHH-based antigen-binding domains have also been prepared (see Figure 2b).

In the chimeric antigen receptors proposed in the present invention, the antigen binding domain contains a Fab fragment or, for example, a monoclonal antibody (see Figure 2a). FabCAR contains two chains: one of which has an antibody-like variable region (VL) and constant region (CL) light chain; and the second has a variable region (VH) and a constant region (CH) of the heavy chain. One chain also contains a transmembrane domain and an intracellular signaling domain. Association between CL and CH results in receptor assembly.

The two Fab CAR chains may have a common structure:

VH - CH - spacer - transmembrane domain - intracellular signaling domain and

VL - CL

or

VL - CL - spacer - transmembrane domain - intracellular signaling domain and

VH - CH

For the chimeric Fab type receptors described herein, the antigen binding domain consists of the VH of one polypeptide chain and the VL of the other polypeptide chain.

Polypeptide chains may contain a linker between the VH/VL domain and the CH/CL domains. The linker can be flexible and provide spatial separation of the VH/VL domain from the CH/CL domains.

Flexible linkers can consist of small non-polar residues such as glycine, threonine and serine. The linker may contain one or more repeats of a glycine-serine linker, such as the (Gly4Ser)n linker (SEQ ID NO: 78), where n denotes the number of repeats. Such or each linker may be less than 50, 40, 30, 20 or 10 amino acids in length.

The present invention also provides cleavable FabCARs that comprise a first antigen binding domain that binds a first target antigen and a second binding domain that binds a second target antigen. Such a cleavable Fab CAR is schematically depicted in Figure 9.

The cleavable Fab CAR may comprise a cleavable linker between the first and second binding domains.

The two chains of a cleavable Fab CAR may have a common structure:

VH1 - VH2 - CH - spacer - transmembrane domain - intracellular signaling domain and

VL1 - VL2 - CL

or

VL1 - CL1 - spacer - transmembrane domain - intracellular signaling domain and

VH2 - CH2

Cleaved FabCAR exhibits reduced accessibility of the membrane-proximal binding domain (binding domain 2 in Figure 9) without cleavage due to steric hindrance due to the first binding domain. Following cleavage, removal of binding domain 1 increases the availability of binding domain 2, allowing it to be activated in the presence of its target antigen.

The cleavable linker may be cleavable by an agent such as a small molecule or an enzyme, such as a protease. The agent can be administered to a patient to cleave the linker and activate or enhance signaling through the second binding domain. Alternatively, the cleavage agent may be naturally expressed in vivo, for example in areas of inflammation or tumor growth/metastasis.

Matrix metalloproteinases (MMPs) are a group of enzymes responsible for the degradation of most extracellular matrix proteins during organogenesis and growth. MMP expression and activity in adult tissues is generally low but is significantly increased in a variety of pathological conditions, including inflammation and cancer.

MMPs are calcium-dependent zinc-containing endopeptidases with a common domain structure. The three common domains are the propeptide, the catalytic domain, and the hemopexin-like C-terminal domain, which is connected to the catalytic domain by a flexible hinge region.

MMPs are present in virtually all types of human cancer; they may be expressed by fibroblasts in the adjacent stroma, cancer-associated fibroblasts, and/or cancer cells other than fibroblasts. Thus, MMPs may influence the tumor environment by promoting angiogenesis, tumor growth, and metastasis. Accordingly, MMP expression is associated with tumor aggressiveness and stage, and with patient prognosis.

Increased MMP expression correlates with increased cancer cell proliferation and increased tumor size. Dysregulation in various types of human cancer has been shown for almost every member of the MMP family, with MMP-1, -2, -7, -9, -13 and -14 being the most commonly affected.

In the cleavable FabCAR proposed in the present invention, the linker between the first and second antigen binding domains can be cleaved by MMP, in particular MMP selected from MMP-1, -2, -7, -9, -13 and -14.

Various sequences are known that are cleaved by MMP enzymes, for example:

MMP-1: PLGLWA (SEQ ID NO: 85)

MMP-2: PAGLAG (SEQ ID NO: 86)

MMP-9: PLGLAG (SEQ ID NO: 87)

The cleavable linker sequence of the cleavable FabCAR of the present invention may comprise one of the sequences shown as SEQ ID NO: 85-87.

CONSTANT AREA DOMAINS

In humans, there are two types of light chains: kappa (κ) chain and lambda (λ) chain. The lambda type includes 4 subtypes: λ1, λ2, λ3 and λ4. The Fab type chimeric receptor light chain constant region can be derived from any of these light chain types.

The light chain constant region of the chimeric receptor of the present invention may have the sequence SEQ ID NO: 1, which is a kappa chain constant domain.

SEQ ID NO: 1

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

There are five types of mammalian immunoglobulin heavy chains: γ, δ, α, μ, and ε, which define the immunoglobulin classes IgG, IgD, IgA, IgM, and IgE, respectively. The γ, δ and α heavy chains have a constant domain consisting of three tandem Ig domains and have a hinge region for added flexibility. The μ and ε heavy chains consist of four domains.

The CH domain of the chimeric Fab type receptor proposed in the present invention may contain the sequence SEQ ID NO: 2, which is derived from the immunoglobulin γ heavy chain.

SEQ ID NO: 2

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

SPACER

A classic CAR contains a spacer sequence to connect the antigen binding domain to the transmembrane domain and to spatially separate the antigen binding domain from the endodomain. A flexible spacer allows the antigen-binding domain to be positioned in different directions to facilitate binding.

In FabCAR (Figure 2a), as in classical chimeric antigen receptor (Figure 2c) and dAbCAR (Figure 2C), a spacer can cause dimerization of two polypeptide chains. Two of the polypeptide chains may contain, for example, one or more cysteine residues to form a disulfide bridge(s). In this regard, the IgG1 hinge region is stable. The IgG1 hinge region spacer may have the sequence set forth as SEQ ID. NO: 3.

SEQ ID NO: 3 (Human IgG1 Hinge Region):

AEPKSPDKTHTCPPCPKDPK

Alternatively, the hinge spacer may have the sequence SEQ ID. NO: 4.

SEQ ID NO: 4 (hinge spacer)

EPKSCDKTHTCPPCP

TRANSMEMBRANE DOMAIN

The transmembrane domain is the part of the chimeric receptor that spans the membrane. The transmembrane domain can have any protein structure that is thermodynamically stable in the membrane. It is typically an alpha helix containing several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the chimeric receptor. Those skilled in the art can determine the presence of a protein's transmembrane domain and the number of times it crosses the membrane using the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/). As an alternative, an artificially engineered transmembrane (TM) domain can be used.

ENDODOMAIN

The endodomain is the signal transducing part of the chimeric receptor. It may be part of or associated with the intracellular domain of the chimeric receptor. After antigen recognition, the receptors are grouped into clusters, native CD45 and CD148 are excluded from the synapse, and the signal is transmitted into the cell. The most commonly used endodomain component is the CD3-zeta endodomain, which contains 3 immunoreceptor tyrosine activating motifs (ITAMs). It transmits an activation signal to the T cell after antigen binding. CD3-zeta may not provide a complete activation signal, so additional co-stimulatory signals may be required. Costimulatory signals stimulate T cell proliferation and survival. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and those belonging to the TNF family (OX40, 41BB, CD27, GITR, etc.). For example, chimeric CD28 and OX40 can be used with CD3-zeta for proliferation/survival signaling, or all three can be used together.

The endodomain may contain:

(i) an endodomain containing an ITAM, such as an endodomain from CD3-zeta; and/or

(ii) a costimulatory endodomain, such as the endodomain from CD28 or ICOS; and/or

(iii) a domain that transmits a survival signal, for example, the endodomain of the TNF receptor family such as OX-40, 4-1BB, CD27 or GITR.

Several systems have been described in which the antigen recognition part is not located on the signal transducing part, but on a separate molecule, such systems are described in WO015/150771; WO2016/124930 and WO2016/030691. Thus, the chimeric receptor proposed in the present invention may contain an antigen binding component comprising an antigen binding domain and a transmembrane domain; which are capable of interacting with a separate intracellular signaling component containing a signaling domain. The vector of the present invention can express a chimeric receptor signaling system comprising such an antigen binding component and an intracellular signaling component.

The chimeric receptor may contain a signal peptide so that when it is expressed within a cell, the resulting protein is directed to the endoplasmic reticulum and subsequently to the surface of the cell in which it is expressed. The signal peptide may be located at the amino terminus of the molecule.

TARGET ANTIGEN

A "target antigen" is a unit that is specifically recognized and bound by the antigen binding domain of the chimeric receptor of the present invention.

The target antigen may be an antigen present on a cancer cell, such as a tumor-associated antigen.

The target antigen for a CAR may be expressed on the target cell at a relatively low density.

The cell of the present invention may have the ability to kill target cells, such as cancer cells, that have a low density of CAR target antigen expression. Examples of known tumor-associated antigens that have low expression density in certain types of cancer include, but are not limited to, ROR1 in CLL, Typr-1 in melanoma, BCMA and TACI in myeloma, and ALK in neuroblastoma.

Example 1 describes a study that examined BCMA expression on melanoma cells. The range of BCMA copy numbers on the melanoma cell surface was found to be low: 348.7–4268.4 BCMA copies per cell, with a mean of 1181 and a median of 1084.9 (Figure 1).

The average copy number of target antigen for a CAR may be less than approximately 10,000; 5000; 3000; 2000; 1000 or 500 copies per target cell.

The average copy number of an antigen on a cell, such as a cancer cell, can be assessed by standard techniques, such as the use of phycoerythrin (PE)-labeled Quantibrite beads, as described in Example 1.

The target antigen for a CAR may be expressed on the target cell with an average copy number of 1500 copies per cell or less, or 1000 copies per cell or less.

The target antigen may, for example, be BCMA, ROR1, Tyrp-1, TACI or ALK.

BCMA

B cell maturation target, also known as BCMA; TR17_HUMAN, TNFRSF17 (UniProt Q02223) is a transmembrane protein that is expressed by mature lymphocytes, such as memory B cells, plasmablasts and bone marrow plasma cells. BCMA is also expressed on myeloma cells. BCMA is a non-glycosylated type III transmembrane protein that is involved in B cell maturation, growth and survival.

The antigen binding domain of a CAR or TanCAR that binds to BCMA can be any domain capable of binding to BCMA. The VH sequences for the VL of fourteen anti-BCMA antibodies are listed below, with the complementarity determining region (CDR) sequences in bold and underlined.

SEQ ID NO: 5 (VH AT 1 vs BCMA)

QIQLVQSGPELVKPGSSVKLSCKTSGFTFSDSYMSWLKQVPGQSIEWIGNIYAGDGATHYHQKFKGKATLTVDTSSSTAYMDLSSLTSEDSALYFCARPLYTTAYYYVGGFAYWGQGTLVTVSS

SEQ ID NO: 6 (VL AT 1 vs BCMA)

DIVMTQSPSSLAVSAGETVTINCKSSQSLLSSGNQKNYLAWYQQKPGQSPKLLIYWASTRQSGVPDRFIGSGSGTDFTLTISSVQAEDLAIYYCQQYYDTPLTFGSGTKLEIK

SEQ ID NO: 7 (VH AT 2 vs BCMA)

EVKLVESGGGLVQPGRSLKLSCTASGTFFSNYDMAWVRQAPTKGLEWVASISTSGDTIYYRDSVKGRFTVSRDKAKSTLYLQMDSLRSEDTATYYCARHDYYDGYQSFAYWGQGTLVTVSS

SEQ ID NO: 8 (VL AT 2 vs BCMA)

NTVMTQSPTSMSISVGDRVTMNCKASQNVGNNIAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGSGSGTDFTLTINSVQAEDAAFYYCQRIYNSALTFGSGTKLEIK

SEQ ID NO: 9 (VH AT 3 vs BCMA)

QVQLQQSGAALVKPGASVKMSCKASGYTFTDYWVSWVKQSHGKSLEWIGEIYPNSGPTNFNKKFKGKATLTVDKSTSTAYMELSRLTSEDSAIYYCTPRTVAPYNWFAYWGQGTLVTVSS

SEQ ID NO: 10 (VL AT 3 vs BCMA)

DIVLTQSPALAVSPGERVSISCRASESVSTRMHWYQQKPGQQPKLLIYGASNLESGVPARFSGSGSGTDFTLTIDPVEADDTATYFCQQSWNDPYTFGAGTKLELK

SEQ ID NO: 11 (VH AT 4 vs BCMA)

EVQLVESGGGLVQPGRSLKLSCSASGFIFSNFDMAWVRQAPRKGLEWVASITTSGGDTHYRDSVKGRFTVSRHNAKSTLYLQMDSLRSEDTATYYCARHVYYGLFWFFDFWGPGTMVTVSS

SEQ ID NO: 12 (VL AT 4 vs BCMA)

NTVMTQSPKSIFISVGDRVTVNCKASQNVGTNVDWYQQKTGQSPKLLIYGASNRYTGVPDRFTGSGSGTDFTFTISNMQAEDLAVYYCMQSNTNPFTFGAGTKLELKR

SEQ ID NO: 13 (VH AT 5 vs BCMA)

EVQLVESGGGLVQPGRSLKLSCTASGTFFSNYDMAWVRQAPTKGLEWVASISTSGDTIYYRDSVKGRFTVSRDKAKSTLYLQMDSLRSEDTATYYCARHDYYDGYQSFAYWGQGTLVTVSS

SEQ ID NO: 14 (VL AT 5 vs BCMA)

DIVMTQSPSTLPASLGERVTISCRASQSISNYLNWYQQKPDGTIKPLIYYTSNLQSGVPSRFSGSGSGTDYSLTISSLEPEDFAMYYCQQDASFPWTFGGGTKLELKR

SEQ ID NO: 15 (VH AT 6 vs BCMA)

EVQLQESGPGLVKPSQSLSLTCSVTGYPITNNYDWSWIRQFPGNKMEWMGYISDSGNTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCASGYISYIPFAFWGQGTLVTVSS

SEQ ID NO: 16 (VL AT 6 vs BCMA)

DIVLTQSPALAVSLGQRATISCRASQSVSISSYNLMQWYQQKPGQQPKLLIYDASNLASGIPARFSGSGSGTDFTLTIDPVQADDIATYYCQQSKDDPNTFGAGTKLEIKR

SEQ ID NO: 17 (VH AT 7 vs BCMA)

EVQLQESGPGLVQPSQTLSLTCSVTGYPITNNYDWSWIRKFPGNKMEWMGYISDSGSTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCASGYISYIPFGFWGQGTLVTVSS

SEQ ID NO: 18 (VL AT 7 vs BCMA)

DIVLTQSPALAVSPGERVTISCRASESVSTRMHWYQQKPGQQPKLLIYGASNLESGVPARFSGSGSGTDFTLTIDPVEADDTATYFCQQSWNDPPTFGSGTKLEIK

SEQ ID NO: 19 (VH AT 8 vs BCMA)

EVQLVESGGGLVQPGRSLKLSCTASGTFFSNYDMAWVRQAPTKGLEWVASISTSGDTIYYRDSVKGRFTVSRDKAKSTLYLQMDSLRSEDTATYYCARHDYYDGYQSFAYWGQGTLVTVSS

SEQ ID NO: 20 (VL AT 8 vs BCMA)

DIVMTQSPASQAVSAGEKVTMSCKSSQSLLYSGDQKNYLAWYQQKPGQSPKLLIYLASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYYCQQHYSYPLTFGSGTKLEIK

SEQ ID NO: 21 (VH AT 9 vs BCMA)

EVQLVESGGGLVQPGRSLKLSCAASGTFFSNYDMAWVRQAPTKGLEWVASISTSGDTIYYRDSVKGRFTVSRDNAKSTLYLQMDSLRSEDTATYYCTRHGYYDGYQSFDYWGQGTLVTVSS

SEQ ID NO: 22 (VL AT 9 vs BCMA)

NTVMTQSPKSMSISVGDRVTMNCKASQNVGNNIAWYQQKPGQSPKLLIYYASNRYTGVPDRFTGGGYGTDFTLTINSVQAEDAATYYCQQWNYPSITFGSGTKLEIK

SEQ ID NO: 23 (VH AT 10 vs BCMA)

EVQLVESGGGLVQPGRSMKLSCAASGTFFSNYDMAWVRQAPTKGLEWVASISPSGGSTYYRDSVKGRFTVSRDNAKSSLYLQMDSLRSEDTATYYCTRGDYGYNYAYWFAYWGQGTLVTVSS

SEQ ID NO: 24 (VL AT 10 vs BCMA)

DIVMTQAPSSMPASLGERVTISCRASQGISNYLNWYQQKPDGTIKPLIYYTSNLQSGVPSRFSGSGSGTDYSLTISSLEPEDFAMYYCQQYDSSPLTFGAGTKLELK

SEQ ID NO: 25 (VH AT 11 vs BCMA)

EVQLVESGGGLVQPGRSLKLSCEASGTFFSNYDMAWVRQAPTKGLEWVASISTSGDSIYYRDSVKGRFTVSRDNVKSTLYLQMDSLRSEDTATYYCARHGYYDGYQSFDYWGQGTLVTVSS

SEQ ID NO: 26 (VL AT 12 vs BCMA)

DIVMTQSPSSLPASLGERVTISCRASQGISNNLNWYQQKPDGTIKPLIYYTSNLQSGVPSRFSGSGSGTDYSLTISSLEPEDFATYYCQQDETFPYTFGAGTKLELK

SEQ ID NO: 27 (VH AT 13 vs BCMA)

EVQLVESGGGLVQPGRSLKLSCAASGTFFSNYDMAWVRQAPTKGLEWVASISPSGGSTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCATHNYYDGSSLFAYWGQGTLVTVSS

SEQ ID NO: 28 (VL AT 13 vs BCMA)

DIVLTQSPALAVSPGERVTISCGANETVSTLVHWYQQKPGQQPKLLIYLASHLESGVPARFSGSGSGTDFTLTIDPVEADDTATYYCQQSWNDPPTFGGGTKLELK

SEQ ID NO: 29 (VH AT 14 vs BCMA)

EVQLVESGGGLVQPGRSLKLSCAASGFIFSDYNMAWVRQAPKKGLEWVATIIYDGSSSTNHGDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCATRPGPFAYWGQGTLVTVS

SEQ ID NO: 30 (VL AT 15 vs BCMA)

DVVLTQTPPTLSATIGQSVSISCRSSQSLLHSNGNTYLHWLLQRPGQSPQFLIYLVSGLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCVHGTHAWTVGGGTKLELK

The BCMA-binding CAR antigen-binding domain may comprise a CDR from any anti-BCMA AT 1-14, as described above.

The BCMA-binding CAR antigen-binding domain may comprise the VH and/or VL sequence from any anti-BCMA Ab 1-14, as described above, or a variant of this sequence that has at least 70, 80, 90, or 90% identity to the sequence , this variant retains the ability to bind to BCMA.

The present invention also provides new BCMA-binding antibodies, designated AT 1-14. The VH, VL and CDR sequences of AT 1-14 are listed in or as SEQ ID NO: 5-30 above.

The BCMA-binding antibody AT 1 demonstrates particularly high efficacy in the CAR format. For example, AT 1 scFv CAR format demonstrated improved target cell killing, IL-2 release, and proliferation compared to various equivalent CARs with alternative BCMA-binding domains (data not provided).

The present invention also provides aspects that are summarized in the numbered paragraphs below.

1. Antigen-binding domain, which contains:

a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences indicated in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29; And

b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the sequences indicated in SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30.

2. Antigen-binding domain, according to paragraph 1, which contains:

a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - GFTFSDSY (SEQ ID NO: 68)

CDR2 - IYAGDGAT (SEQ ID NO: 69)

CDR3 - ARPLYTTAYYYVGGFAY (SEQ ID NO: 70) and

b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - QSLLSSGNQKNY (SEQ ID NO: 71)

CDR2 - WAS (SEQ ID NO: 72)

CDR3 - QQYYDTPLT (SEQ ID NO: 73)

3. Antigen-binding domain, according to paragraph 1, which contains:

a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - GFIFSDYN (SEQ ID NO: 79)

CDR2 - IIYDGSST (SEQ ID NO: 80)

CDR3 - ATRPGPFAY (SEQ ID NO: 81) and

b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - QSLLHSNGNTY (SEQ ID NO: 82)

CDR2 - LVS (SEQ ID NO: 83)

CDR3 - VHGTHAWT (SEQ ID NO: 84)

4. The antigen binding domain according to paragraph 1, which contains a VH domain having one of the sequences SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29; and a VL domain having one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30.

5. The antigen binding domain according to paragraph 3, which contains a VH domain having the sequence SEQ ID NO: 5; and a VL domain having the sequence SEQ ID NO: 6.

6. The antigen binding domain according to paragraph 3, which contains a VH domain having the sequence SEQ ID NO: 29; and a VL domain having the sequence SEQ ID NO: 30.

7. An antibody that contains an antigen binding domain according to any of the previous paragraphs.

8. An antibody-drug conjugate (ADC) or bispecific T-cell recruiter (BiTE) that contains an antibody according to paragraph 7.

9. A chimeric antigen receptor (CAR) that contains an antigen binding domain according to any of paragraphs 1-6.

10. CAR according to paragraph 9, which is FabCAR.

11. A nucleic acid sequence that encodes an antigen binding domain according to any of paragraphs 1-6, and an antibody according to paragraph 6, an ADC or BiTE according to paragraph 8, or a CAR according to paragraph 9 or 10.

12. A vector that contains the nucleic acid sequence according to paragraph 11.

13. A cell that expresses CAR according to paragraph 9 or 10.

14. The method of obtaining a cell according to paragraph 13, which includes the step of introducing the CAR coding nucleic acid sequence according to paragraph 11 into the cell.

15. A pharmaceutical composition which contains a plurality of cells according to paragraph 13 together with a pharmaceutically acceptable carrier, diluent or excipient.

16. A method of treating cancer, which includes the step of administering to a subject a pharmaceutical composition according to paragraph 15.

17. The method according to paragraph 15, where the cancer is B-cell leukemia or lymphoma.

18. Cell according to paragraph 13 for use in the treatment of cancer.

19. Use of a cell according to paragraph 13 in the production of a pharmaceutical composition for the treatment of cancer.

The general characteristics of, for example, chimeric antigen receptors, nucleic acid sequences and constructs, vectors, cells, pharmaceutical compositions, and methods for preparing and using cells described in the preceding and following sections also apply to the corresponding components described in the above paragraphs.

ROR1

Orphan receptor type receptor tyrosine kinase-1 (ROR1), also known as neurotrophic receptor tyrosine kinase 1 (NTRKR1), is a receptor tyrosine kinase that modulates axonal growth in the central nervous system. It is a type I membrane protein and belongs to the ROR subfamily of cell surface receptors.

Expression of ROR1 has been shown in ovarian cancer stem cells, in which it appears to play a functional role in promoting migration/invasion or spheroid formation in vitro and tumor engraftment in immunodeficient mice. Treatment with humanized mAbs specific for ROR1 can inhibit the ability of ovarian cancer cells to migrate, form spheroids, or engraft in immunodeficient mice.

WO2017/072361 describes several ROR1-specific antibodies suitable for use in the development of CARs.

TACI

Transmembrane activator and calcium modulator cyclophilin ligand (CAML) partner TACI (UniProtKB: O14836) is a regulator of immune responses and, like BCMA, is predominantly expressed by mature lymphocytes such as CD27+ memory B cells, particularly marginal zone B cells , bone marrow plasma cells and myeloma cells.

In addition, TACI is expressed on macrophages and mediates macrophage survival. TACI is a lymphocyte-specific member of the tumor necrosis factor receptor superfamily (TNF), also known as tumor necrosis factor receptor superfamily member 13B (TNFRSF13B), and can be shed from the cell surface and circulate in a soluble form. In contrast to BCMA, TACI is virtually absent from germinal center B cells. TACI is known to function as a receptor for TNFSF13/APRIL and TNFSF13B/BAFF and binds to both ligands with high affinity. TACI inhibits B cell expansion and stimulates plasma cell differentiation and survival.

TACI is a member of the tumor necrosis factor receptor (TNFR) superfamily and serves as a key regulator of B cell function. TACI binds with high affinity to two ligands, APRIL and BAFF, and its extracellular region contains two cysteine-rich domains (CRDs). However, another form of TACI exists in which the CRD at the N-terminus is removed by alternative splicing. It was shown that this shorter form is capable of ligand-induced signal transduction, and that the second CRD (TACI_d2) itself has full affinity for both ligands (Hymowitz et al 2005 Am Soc. Biochem. And Mol. Biol. Inc 280(8) 7218 -7227).

The TACI_d2 crystal structure was solved simultaneously with the cocrystal structure of the APRILTACI_d2 and APRIL BCMA complexes (Hymowitz et al. 2005, as above).

The TACI CRD and the other TNFR CRDs share a common sequence feature, the so-called DXL motif, which consists of a conserved 6-residue sequence (Phe/Tyr/Trp)-Asp-Xaa-Leu-(Val/Thr)-(Arg/Gly) ( SEQ ID NO: 74). This motif is required for APRIL or BAFF binding. The receptor motif binds within a hydrophobic pocket and interacts with two conserved arginine residues on the surface of BAFF.

The antigen binding domain of a CAR or tanCAR that binds to TACI can be any domain capable of binding to TACI. For example, the antigen binding domain may comprise a TACI binding element derived from one of the commercially available anti-TACI antibodies listed in Table 1.

Table 1

AT vs TACI Company 1A1 BioLegend ab5994 Abcam Ab79023 Abcam 11H3 Affymetrix eBioscience

The antigen binding domain may comprise one of the TACI binding elements of clones 2H6, 2G2, 1G6 or 4B11.

These TACI binding elements have the following sequences:

2H6

ScFv:EVQLQQSGPELVKPGASVRMSCKASGYTFTNYVMHWVKQKPGQGLEWIGYINPSNDDTKYTEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGTHGDYYALDYWGQGTSVTVSSGGGGAGGGGSGGGGSDIVLTQSPASLAVSLGQSVTISCRASESVEYYGTSLMQWYQQKPGQAPKLLI YGASNVESGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKVPWTFGGGTKLEIKR (SEQ ID NO: 31)

CDR H1: NYVMH (SEQ ID NO: 32)

CDR H2: YINPSNDDTKYTEKFKG (SEQ ID NO: 33)

CDR H3: GTHGDYYALDY (SEQ ID NO: 34)

CDR L1: RASESVEYYGTSLMQ (SEQ ID NO: 35)

CDR L2: GASNVES (SEQ ID NO: 36)

CDR L3: QQSRKVP (SEQ ID NO: 37)

2G2

ScFv:QVTLKESGPGMLQPSQTLSLTCSFSGFSLSTFGMGVGWIRQPSGKGLEWLAHIWWDDAQYSNPALRSRLTISKDTSKNQVFLKIANVDTADTATYYCSRIHSYYSYDEGFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSQKFMSTTVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSASN RYTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSSYRTFGGGTKLEIKR (SEQ ID No. 38)

CDR H1: TFGMGVG (SEQ ID NO: 39)

CDR H2: HIWWDDAQYSNPALRS (SEQ ID NO: 40)

CDR H3: RIHSYYSYDEGFAY (SEQ ID NO: 41)

CDR L1: KASQNVGTAVA (SEQ ID NO: 42)

CDR L2: SASNRYT (SEQ ID NO: 43)

CDR L3: QQYSSY (SEQ ID NO: 44)

1G6

VH:QVQLKQSGPGLVAPSQSLSITCTVSGFSLTSYGVDWVRQSPGKGLEWLGIIWGGGRTNYNSAFKSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCASGDRAADYWGQGTSVTVSS (SEQ ID No. 45)

CDR H1: SYGVD (SEQ ID NO: 47)

CDR H2: IIWGGGRTNYNSAFKS (SEQ ID NO: 48)

CDR H3: GDRAADY (SEQ ID NO: 49)

VL:DIVMTQSQKFMSTTVGDRVTITCKASQNVGTAVAWYQQKPGQSPKLLIYSASNRYTGVPVRFTGSGSGTDFTLTINNMQSEDLADYFCQQYSSYPLTFGAGTKLELK (SEQ ID No. 46)

CDR L1: KASQNVGTAVA (SEQ ID NO: 50)

CDR L2: SASNRYT (SEQ ID NO: 51)

CDR L3: QQYSSYP (SEQ ID NO: 52)

4B11

VH:EVQLQQSVAELVRPGASVKLSCTASGFNIKNTYIHWVKQRPEQGLEWIGKIDPANGNSEYAPKFQGKATITADTSSNTAYLQLSSLTSEDTTIYYCTSGYGAYWGQGTTLTVSS (SEQ ID NO: 53)

CDR H1: NTYIH (SEQ ID NO: 55)

CDR H2: KIDPANGNSEYAPKFQG (SEQ ID NO: 56)

CDR H3: GYGAY (SEQ ID NO: 57)

VL:DIVLSQSPSSLAVSIGEKVTLSCKSSQSLLYSSNQKNYLAWFQQKPGQSLKLLIYWASTREFGVPDRFTGSGSGTDFTLTISSVKTEDLAVYYCQQYYTWTFGGGTKLEIK (SEQ ID NO: 54)

CDR L1: KSSQSLLYSSNQKNYLA (SEQ ID NO: 58)

CDR L2: WASTREF (SEQ ID NO: 59)

CDR L3: QQYYTW (SEQ ID NO: 60)

The present invention provides an anti-TACI agent, such as an antibody, scFv, CAR or tanCAR, which contains a TACI-binding domain based on the domain of one of the clones: 2H6, 2G2, 1G6 or 4B11. The anti-TACI agent may contain one or more CDRs with the sequences SEQ ID NO: 32-37; 39-44; 47-52; 55-60. The agent may contain one of the following groups of six CDRs: SEQ ID NO: 32-37; SEQ ID NO: 39-44; SEQ ID NO: 47-52 and 55-60.

The anti-TACI agent may contain VH and/or VL sequences of SEQ ID NO: 31 or 38 (VH located before the serine-glycine linker, VL located after the serine-glycine linker), for example, in Fab format. The anti-TACI agent may contain the VH sequence SEQ ID NO: 45 or 53 and/or the VL sequence SEQ ID NO: 46 or 54, for example, in Fab format. The anti-TACI agent may comprise a scFv represented by SEQ ID NO: 31 or 38 or a scFv formed by linking SEQ ID NO: 45 to SEQ ID NO: 46 or SEQ ID NO: 53 to SEQ ID NO: 54.

For all of the sequences listed above, the anti-TACI agent may contain a sequence having 80, 85, 90, 95 or 98% identity to the specified sequence, provided that the sequence variant retains the ability to bind to TACI.

Tyrp-1

Tyrp1 is a melanocyte-specific gene product involved in melanin synthesis. While mouse Tyrp1 has dihydroxyindole carboxylic acid oxidase activity, its function in human melanocytes is less clear. In addition to its role in melanin synthesis, Tyrp1 is involved in stabilizing protein tyrosinase and modulating its catalytic activity. Tyrp1 is also involved in the maintenance of melanosome structure and influences the proliferation and death of melanocytes.

WO2009114585 describes several antibodies that bind to Tyrp-1. Antibodies against Tyrp-1 are also commercially available, such as SAB1406566 and HPA000937 from Sigma-Aldrich.

ALK

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. The protein includes an extracellular domain, a hydrophobic extended region corresponding to the transmembrane region that passes once through the membrane, and an intracellular kinase domain. Human ALK sequences are publicly available, for example GENBANK® accession numbers: NP_004295 (protein) and NM_004304 (nucleic acid), and UniProt Acc.: No Q9UM73.

WO2015069922 describes several scFv-type antigen-binding domains that bind to ALK.

Target antigens for cleaved FabCAR

The Fab CAR proposed in the present invention may have two antigen binding domains, as schematically depicted in Figure 9.

In the cleavable Fab CAR of the present invention, the first and/or second binding domain can bind to an antigen that is expressed at a relatively low density on the target cell. For example, it can bind to an antigen selected from ROR1, Typr-1, BCMA, TACI and ALK. In particular, the target antigen for the second (membrane proximal) binding domain may be an antigen expressed at a relatively low density on the target cell.

Binding of the target antigen to the first (membrane distal) binding domain can be used to target the CAR to the tumor. Cleavage of the linker in the tumor allows activation by binding of a second antigen-binding domain to a second target antigen, resulting in destruction of the tumor cell. However, it should be noted that binding of the first target antigen by the first binding domain can also trigger cell killing, but the killing efficiency may be reduced (compared to killing induced by binding of the target antigen by the second binding domain) due to ineffective synapse formation.

One advantage of cleavable FabCAR is that the internal binding domain (ie, the second binding domain) in the intact molecule is partially hidden due to steric hindrance. This may improve safety in cases where the second target antigen is expressed to some extent in one or more healthy tissues, since the exposure of the second binding domain to its target antigen will be reduced before the first binding point is removed, which cannot occur before the expression The CAR cell will reach the tumor.

Therefore, the second antigen may be expressed at high density on tumor cells and at low density on cells of one or more healthy tissues.

The target antigen for the first or second binding domains may be selected from one of the following: ErbB2, MUC1, CD33, CD123, PSMA, EpCAM, GD2, NCAM, folate binding protein and MUC16.

In particular, the cleavable FabCAR of the present invention may contain binding domains directed against one of the following pairs of target antigens: ErbB2 and MUC1; CD33 and CD123; PSMA and EpCAM; GD2 and NCAM; folate binding protein and MUC16. In these target antigen pairs, binding domain 1 and binding domain 2 can bind to either antigen, for example, in a target antigen pair ErbB2 and MUC1, binding domain 1 can bind to ErbB2 and binding domain 2 can bind to MUC1, or the binding domain 2 can bind to ErbB2, and binding domain 1 can bind to MUC1.

In particular, the cleavable FabCAR of the present invention may contain binding domains directed against one of the pairs of target antigens listed in Table 2.

table 2

Indication Antigen target for binding domain 1 Antigen target for binding domain 2 Mammary cancer ErbB2 MUC1 AML CD33 CD123 Prostate cancer PSMA EpCAM Neuroblastoma GD2 NCAM Ovarian cancer Folate binding protein MUC16

LOGIC GATES "OR"

The CARs of the present invention can be used in combination with one or more other activating or inhibitory chimeric antigen receptors. For example, they can be used in combination with one or more other CARs as part of a “gate”—a combination of CARs that, when expressed in a cell, such as a T cell, is capable of detecting a specific expression pattern of at least two target antigens. If at least two target antigens are arbitrarily designated as antigen A and antigen B, the three possibilities are as follows:

“OR ELEMENT” - the T cell is activated when antigen A or antigen B is present on the target cell

“ELEMENT AND” - The T cell is activated only when both antigens A and B are present on the target cell

“NAND ELEMENT” - the T cell is activated if only antigen A is present on the target cell, but is not activated if both antigens A and B are present on the target cell

Engineered T cells expressing these CAR combinations can be tailored to be uniquely specific to cancer cells based on their characteristic expression (or lack of expression) of two or more markers.

Such "logical elements" are described, for example, in WO2015/075469, WO2015/075470 and WO2015/075470.

An “OR element” contains two or more activating CARs, each of which is specific for a different target antigen expressed by the target cell. The advantage of the OR element is that the effective amount of the target antigen on the target cell is increased due to the fact that it is actually a combination of antigen A + antigen B. This is especially important for antigens expressed on the target cell at variable or low densities, as the density of a single antigen may be below the threshold required for effective CAR T cell targeting. It also avoids the phenomenon of antigen escape. For example, some lymphomas and leukemias become CD19 negative after targeting CD19: the use of an OR element that targets CD19 in combination with another antigen provides a “spare” antigen in case of such a situation.

The FabCAR of the present invention can be used in an OR element in combination with a second CAR against a second target antigen expressed by the target cell.

For a FabCAR against BCMA, the OR element may comprise a CAR against a second antigen expressed by B cells or plasma cells, such as CD19, TACI or FcRL5.

The second CAR may have any suitable antigen-binding domain, for example, a scFv-based, domain-specific antibody (dAb) or Fab-based binding domain.

The OR gate may be a double FabCAR. In this regard, a cell may express two separate CARs with Fab-type antigen-binding domains. FabCARs can be directed against, for example, BCMA and TACI or BCMA and FcRL5.

The present invention also provides a ternary OR gate containing three CARs, at least one of which is a FabCAR according to the first aspect of the present invention. The ternary OR element may, for example, comprise BCMA FabCAR, CD19 CAR and FcRL5 CAR or FabCAR.

The second CAR may contain a spacer to spatially separate the antigen binding domain from the transmembrane domain and provide a degree of flexibility. A variety of sequences are widely used as spacers for CARs, such as the Ig G1 Fc region, the IgG1 hinge region (as described above), or the human CD8 stem. The spacer may contain a supercoiled domain, for example as described in WO2016/151315.

The second CAR contains an activating endodomain. It may, for example, contain an endodomain from CD3ζ. It may contain one or more co-stimulatory domains as described above. For example, it may contain endodomains from CD28, OX-40 or 4-1BB.

CD19 BINDING ELEMENTS

Several anti-CD19 antibodies have been previously described in CAR format, such as fmc63, 4G7, SJ25C1, CAT19 (as described in WO2016/139487) and CD19ALAb (as described in WO2016/102965).

The anti-CD19 CAR for use in the dual or triple OR element of the present invention may comprise an antigen binding domain, such as a scFv-type antigen binding domain derived from one of these anti-CD19 antibodies.

The CD19 binding domain may contain:

a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences:

CDR1 - GYAFSSS (SEQ ID NO: 61);

CDR2 - YPGDED (SEQ ID NO: 62)

CDR3 - SLLYGDYLDY (SEQ ID NO: 63) and

b) a light chain variable region (VL) having a CDR with the following sequences:

CDR1 - SASSSVSYMH (SEQ ID NO: 64);

CDR2 - DTSKLAS (SEQ ID NO: 65)

CDR3 - QQWNINPLT (SEQ ID NO: 66).

It is possible to introduce one or more mutations (substitutions, additions or deletions) in each CDR without negatively affecting CD19 binding ability. Each CDR may, for example, contain one, two or three amino acid mutations.

CDRs can be in the format of a single chain variable fragment (scFv), which is a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody connected by a short linker peptide of 10 to about 25 amino acids in length. scFv can have a VH-VL direction, that is, the VH is located at the amino terminus of the CAR molecule, and the VL domain is connected to the spacer and in series with the transmembrane domain and endodomain.

CDRs can be inserted (grafted) into a human antibody or scFv framework. For example, a CAR of the present invention may comprise a CD19 binding domain consisting of or containing one of the following sequences.

The anti-CD19 CAR may contain the following VH sequence:

SEQ ID NO: 67 - VH sequence from mouse monoclonal antibody

QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSS

The anti-CD19 CAR may contain the following VL sequence:

SEQ ID NO: 75 - VL sequence from mouse monoclonal antibody

QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFSGSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR

The anti-CD19 CAR may contain the following scFv sequence:

SEQ ID NO: 76 - VH-VL scFv sequence from mouse monoclonal antibody

QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDEDTNYSGKFKDKATLTADKSSTTAYMQLSSLTSEDSAVYFCARSLLYGDYLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPDRFS GSGSGTSYFLTINNMEAEDAATYYCQQWNINPLTFGAGTKLELKR.

The percentage identity of two polypeptide sequences can be easily determined using programs such as BLAST, which is freely available at: http://blast.ncbi.nlm.nih.gov.

FCRL5

Fc receptor-like protein 5 (FcRL5) is a member of the immunoglobulin receptor superfamily and the Fc receptor-like protein family. FcRL5 is a type I transmembrane protein that spans the membrane once and contains 8 C2 type immunoglobulin-like domains. The mass of the mature protein is 106 kDa.

FCRL5 has a cytoplasmic tail with two inhibitory ITIM motifs that transmit signals through phosphorylation. It inhibits B cell antigen receptor signaling by recruiting SHP1 after B cell antigen receptor costimulation, resulting in decreased calcium influx and phosphorylation of its constituent protein tyrosine. Costimulation of FCRL5 and B cell antigen receptor stimulates the proliferation and differentiation of naïve B cells. FCRL5 is expressed on mature B cells and plasma cells, and its expression is induced by Epstein-Barr virus (EBV) proteins. Its overexpression is characteristic of malignant B cells from patients with hairy cell leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma.

Commercially available antibodies against FcRL5 are known, such as CD307e (ThermoFisher) and REA391 (Miltenyi Biotec).

WO2016090337 describes several scFv-type antigen-binding domains that bind to FcRL5.

In the OR element of the present invention, the anti-FcRL5 CAR may comprise a dAb, scFv or Fab based antigen binding domain. FcRL5 may contain a Fab-based antigen binding domain.

CONSTRUCTION OF NUCLEIC ACID

The present invention also provides a nucleic acid construct encoding the chimeric receptor of the present invention.

The nucleic acid construct encoding FabCAR (Figure 2a) may have the structure:

VH-CH-spacer-TM-endo-coexpr-VL-CL or

VL-CL-spacer-TM-endo-coexpr-VH-CH

Where:

VH is a nucleic acid sequence encoding a heavy chain variable region;

CH is a nucleic acid sequence encoding a heavy chain constant region;

spacer is a nucleic acid encoding a spacer;

TM is a nucleic acid sequence encoding a transmembrane domain;

endo is a nucleic acid sequence encoding an endodomain;

coexpr is a nucleic acid sequence allowing co-expression of the first and second polypeptides;

VL is a nucleic acid sequence encoding a light chain variable region; And

CL is a nucleic acid sequence encoding a light chain constant region.

With the above two structures, the nucleic acid sequences encoding the two polypeptides can be arranged in any order in the construct.

Also provided is a nucleic acid construct encoding an OR element that contains two or more CARs, at least one of which is a FabCAR according to the present invention.

A nucleic acid construct encoding a double OR element may have the structure:

VH-CH-spacer1-TM1-endo1-coexpr1-VL-CL-coexpr2-AgBD-spacer2-TM2-endo2; or

VL-CL-spacer-TM1-endo1-coexpr1-VH-CH-coexpr2-AgBD-spacer2-TM2-endo2,

Where:

VH is a nucleic acid sequence encoding the heavy chain variable region of the first CAR;

CH is a nucleic acid sequence encoding the heavy chain constant region of the first CAR;

Spacer 1 is a nucleic acid sequence encoding the spacer of the first CAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

Endo1 is a nucleic acid sequence encoding the endodomain of the first CAR;

Coexpr1 and coexpr2, which may be the same or different, are nucleic acid sequences allowing for co-expression of the first and second polypeptides of the first CAR; as well as the first and second CAR;

VL is a nucleic acid sequence encoding the light chain variable region of the first CAR;

CL is a nucleic acid sequence encoding the light chain constant region of the first CAR;

AgBD is a nucleic acid sequence encoding the antigen binding domain of the second CAR;

Spacer2 is a nucleic acid sequence encoding a second CAR spacer;

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR; And

Endo2 is a nucleic acid sequence encoding the endodomain of a second CAR.

The antigen binding domain of the second CAR may, for example, be a scFv or a dAb.

In both of the above structures, the nucleic acid sequences encoding the two polypeptides of the first CAR and the nucleic acid sequences encoding the first and second CARs may be arranged in any order in the construct.

The nucleic acid construct encoding the double FabCAR OR element may have the structure:

VH1-CH1-spacer1-TM1-endo1-coexpr1-VL1-CL1-coexpr2-VH2-CH2-spacer2-TM2-endo2-coexpr3-VL2-CL2;

VH1-CH1-spacer1-TM1-endo1-coexpr1-VL1-CL1-coexpr2-VL2-CL2-spacer2-TM2-endo2-coexpr3-VH2-CH2;

VL1-CL1-spacer1-TM1-endo1-coexpr1-VH1-CH1-coexpr2-VL2-CL2-spacer2-TM2-endo2-coexpr3-VH2-CH2 or

VL1-CL1-spacer1-TM1-endo1-coexpr1-VH1-CH1-coexpr2-VH2-CH2-spacer2-TM2-endo2-coexpr3-VL2-CL2;

Where:

VH1 is a nucleic acid sequence encoding the heavy chain variable region of the first CAR;

CH1 is a nucleic acid sequence encoding the heavy chain constant region of the first CAR;

Spacer1 is the nucleic acid sequence encoding the spacer of the first CAR;

TM1 is a nucleic acid sequence encoding the transmembrane domain of the first CAR;

Endo1 is a nucleic acid sequence encoding the endodomain of the first CAR;

Coexpr1, coexpr2 and coexpr 3, which may be the same or different, are nucleic acid sequences allowing for co-expression of the first and second polypeptides of the first CAR; and first and second polypeptides of the second CAR;

VL2 is a nucleic acid sequence encoding the light chain variable region of a second CAR;

CL2 is a nucleic acid sequence encoding the light chain constant region of a second CAR;

VH2 is a nucleic acid sequence encoding the heavy chain variable region of a second CAR;

CH2 is a nucleic acid sequence encoding the heavy chain constant region of the second CAR;

Spacer 2 is a nucleic acid sequence encoding a second CAR spacer;

TM2 is a nucleic acid sequence encoding the transmembrane domain of the second CAR;

Endo2 is a nucleic acid sequence encoding the endodomain of a second CAR;

VL2 is a nucleic acid sequence encoding the light chain variable region of a second CAR;

CL2 is a nucleic acid sequence encoding the light chain constant region of a second CAR;

In this application, the terms "polynucleotide", "nucleotide" and "nucleic acid" are used synonymously.

One skilled in the art will appreciate that due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it should be understood that those skilled in the art can routinely make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage frequency of any particular host organism. in which polypeptides are expected to be expressed.

Nucleic acids according to the present invention may be part of DNA or RNA. They can be single-stranded or double-stranded. They may also be polynucleotides, which include synthetic or modified nucleotides. Several different types of nucleotide modifications are known in the art. These include methylphosphonate and phosphorothioate variants of the backbone, the addition of an acridine or polylysine chain at the 3' and/or 5' ends of the molecule. For use as described herein, it should be understood that the polynucleotides may be modified by any of the methods available in the art. Such modifications can be made to increase the activity or lifespan of polypeptides of interest in vivo.

The terms “variant,” “homolog,” or “derivative” with respect to a nucleotide sequence include any substitution, variation, modification, substitution, deletion, or addition of one (or more) nucleic acid from or to the sequence.

In the above construct, "coexpr" is a nucleic acid sequence that allows the two polypeptides to be co-expressed as separate units. This may be a sequence encoding a cleavage site that allows the nucleic acid construct to produce both polypeptides linked by the cleavage site(s). The cleavage site may be self-cleaving, such that once a polypeptide is synthesized it is immediately cleaved into individual peptides without the need for external cleavage.

The cleavage site can be any sequence that allows two polypeptides to separate.

In this application, the term “cleavage” is used for convenience, whereas the cleavage site may cause the peptides to be separated into separate units through a mechanism other than classical cleavage. For example, several models have been proposed to explain the “cleaving” activity of foot-and-mouth disease virus (FMDV) self-cleaving peptide 2A (see below): proteolysis by a host cell proteinase, autoproteolysis, or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82 :1027-1041). The exact mechanism of such "cleavage" is not important for the purpose of the present invention, provided that the cleavage site, if located between the nucleic acid sequences that encode proteins, allows the proteins to be expressed as separate units.

The cleavage site may, for example, be a furin cleavage site, a tobacco etch virus (TEV) cleavage site, or encode a self-cleaving peptide.

“Self-cleaving peptide” means a peptide that functions such that, upon synthesis of a polypeptide comprising proteins and a self-cleaving peptide, it instantly “cleaves” or separates into separate or individual first and second peptides without the need for external cleavage action.

The self-cleaving peptide may be the 2A self-cleaving peptide of aphtho- or cardioviruses. Primary 2A/2B cleavage of aphtho- or cardioviruses is mediated by “cleavage” of 2A at its own C-terminus. In aphthoviruses such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus, region 2A is a short fragment of approximately 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), forms an autonomous element capable of mediating “cleavage” at its own C-terminus (Donelly et al. (2001), as above).

"2A-like" sequences have been identified in picornaviruses other than aphtho- and cardioviruses, "picorna-like" insect viruses, type C rotaviruses and repeat sequences of the trypanosome genus and bacterial sequence (Donelly et al. (2001), as above) .

The cleavage site may contain a 2A-like sequence shown in SEQ ID NO: 77 (RAEGRGSLLTCGDVEENPGP).

CHIMERIC CYTOKINE RECEPTOR

The nucleic acid construct of the present invention may also comprise one or more nucleic acid sequence(s) encoding a chimeric cytokine receptor.

Chimeric cytokine receptors are described in WO2017/029512, which is incorporated herein by reference.

The chimeric cytokine receptor may contain:

an exodomain that binds the ligand, and

cytokine receptor endodomain.

The ligand may be, for example, a tumor secreted factor, a chemokine, or a cell surface antigen.

The chimeric cytokine receptor contains two polypeptides:

(i) the first polypeptide which contains:

(a) the first antigen-binding domain, which binds the first epitope of the ligand

(b) the first chain of the cytokine receptor endodomain and

(ii) a second polypeptide which contains:

(a) a second antigen-binding domain that binds to a second ligand epitope (b) a second strand of the cytokine receptor endodomain.

In an alternative embodiment, the chimeric cytokine receptor may comprise a dimerization domain and a cytokine receptor endodomain.

Dimerization can occur spontaneously, in which case the chimeric transmembrane protein will be constitutively active. Alternatively, dimerization may occur only in the presence of a chemical dimerization inducer (CID), in which case the transmembrane protein triggers cytokine-type signaling only in the presence of the CID.

Suitable dimerization domains and CIDs are described in WO2015/150771, the materials of which are incorporated herein by reference.

For example, one dimerization domain may contain the rapamycin-binding domain of FK-binding protein 12 (FKBP12), another may contain the FKBP12-rapamycin-binding (FRB) domain of mTOR; and the CID may be rapamycin or a derivative thereof.

In cases where the dimerization domain spontaneously heterodimerizes, it may be based on the antibody dimerization domain. In particular, it may comprise a dimerization portion of a heavy chain constant domain (CH) and a constant light chain domain (CL). The "dimerization portion" of the constant domain is the portion of the sequence that forms the disulfide bond between the chains.

The chimeric cytokine receptor may contain the Fab portion of the antibody as an exodomain, for example, as schematically depicted in Figure 4.

A chimeric cytokine receptor may contain two polypeptides:

(i) the first polypeptide which contains:

(a) the first dimerization domain and

(b) the first chain of the cytokine receptor endodomain and

(ii) a second polypeptide which contains:

(a) a second dimerization domain that dimerizes with the first dimerization domain, and

(b) the second chain of the cytokine receptor endodomain.

A chimeric cytokine receptor may contain two polypeptides:

(i) the first polypeptide which contains:

(a) heavy chain constant domain (CH)

(b) the first chain of the cytokine receptor endodomain and

(ii) a second polypeptide which contains:

(a) light chain constant domain (CL)

(b) the second chain of the cytokine receptor endodomain.

The cytokine receptor endodomain may, for example, contain:

(i) IL-2 receptor β-chain endodomain

(ii) endodomain of the IL-7 receptor α-chain or

(iii) IL-15 receptor α-chain endodomain and/or

(iv) the common endodomain of the receptor γ chain.

A nucleic acid construct encoding a constitutively active chimeric Fab-type cytokine receptor may have the following general structure:

CH-CRE1-coexpr-CL-CRE2 or

CL-CRE2-coexpr-CH-CRE1

Where:

CH is a nucleic acid sequence encoding the heavy chain constant region of the first polypeptide;

CRE1 is a nucleic acid sequence encoding the first strand of the endodomain of a cytokine receptor of a first polypeptide;

coexpr is a sequence allowing co-expression of the first and second polypeptides;

CH is a nucleic acid sequence encoding a heavy chain constant region in the second polypeptide;

CRE2 is a nucleic acid sequence encoding the second strand of a cytokine receptor endodomain in a second polypeptide;

The nucleic acid construct of the present invention may encode a FabCAR and a chimeric cytokine receptor.

VECTOR

The present invention also provides a vector or set of vectors that contains one or more nucleic acid sequences encoding a chimeric receptor according to the present invention. Such a vector can be used to introduce nucleic acid sequence(s) into a host cell such that it expresses a CAR according to the first aspect of the present invention.

A vector is also provided containing a nucleic acid construct from nucleic acid sequences encoding a FabCAR and a chimeric cytokine receptor.

The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector, a lentiviral vector, a transposon vector, or a synthetic mRNA vector.

The vector may be capable of transfecting or transducing a cell, such as a T cell or NK cell.

CELL

The present invention also provides a cell that contains the chimeric antigen receptor proposed in the present invention. A cell may contain two or more CARs, for example it may contain a double or triple OR element as described above.

Also provided is a cell that co-expresses a FabCAR or OR element of the present invention and a chimeric cytokine receptor.

The cell may contain a nucleic acid or vector as provided in the present invention.

The cell may be a cytolytic immune cell such as a T cell or NK cell.

T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes such as B cells and natural killer cells (NK cells) by the presence of the T cell receptor (TCR) on the cell surface. There are different types of T cells, a summary of which is provided below.

T helper cells (Tx cells) assist other white blood cells in immunological processes, including the maturation of B lymphocytes into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. Tx cells express CD4 on their surface. Tx cells become activated after the presentation of peptide antigens to them by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including Th1, Th2, Th3, Th17, Th9, or follicular T helper (Ftx) cells, which secrete different cytokines to enhance different types of immune responses.

Cytolytic T cells (or cytotoxic T cells, CTL) destroy virally infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Under the influence of IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into anergic state, which prevents the development of autoimmune diseases such as experimental autoimmune encephalomyelitis.

Memory T cells are a subpopulation of antigen-specific T cells that persist in the body for a long time after the infection has resolved. Upon repeated exposure to the antigen they cognate, their rapid expansion occurs with the appearance of a large number of effector T cells, which provides the immune system with a “memory” of past infections. Memory T cells come in three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be CD4+ or CD8+. Memory T cells typically express CD45RO protein on their surface.

Regulatory T cells (Treg cells), formerly known as suppressor T cells, are essential for maintaining immunological tolerance. Their main function is to suppress T cell-mediated immunity towards completion of the immune response and reduce the activity of autoreactive T cells that have escaped the process of negative selection in the thymus.

Two main classes of CD4+ Treg cells have been described: natural Treg cells and adaptive Treg cells.

Natural Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) appear in the thymus and have been associated with interactions between developing T cells and myeloid (CD11c+) as well as plasmacytoid (CD123+) dendritic cells that have been activated by thymic stromal cells. lymphopoietin (TSLP). Natural Tregs can be distinguished from other T cells by the presence of the intracellular molecule FoxP3. Mutations in the FoxP3 gene can prevent the development of regulatory T cells, which causes the fatal autoimmune disease X-linked syndrome of immune dysregulation, polyendocrinopathy and enteropathy (IPEX).

Adaptive Treg cells (also known as Treg1 or Treg3) can appear as part of a normal immune response.

The cell may be a natural killer cell (or NK cell). NK cells form part of the innate immune system. NK cells mediate rapid responses to characteristic signals of virus-infected cells in an MHC-independent manner.

NK cells (belonging to the group of lymphoid cells of the innate immune system) are defined as large granular lymphocytes (LGLs) and are the third cell type differentiated from the common lymphoid precursor that gives rise to B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils and thymus, from where they are released into the bloodstream.

The cells proposed in the present invention may be any of the types of cells mentioned above.

Cells according to the present invention can be obtained either ex vivo from the peripheral blood of the patient himself (1st party), or by transplantation of hematopoietic stem cells from the peripheral blood of a donor (2nd party), or from the peripheral blood of a third party donor (3rd party ).

Alternatively, the cells may be derived from differentiation of inducible progenitor cells or embryonic progenitor cells ex vivo, for example, into T or NK cells. Alternatively, an immortalized T cell line that has retained its lytic function and can act as a drug can be used.

In all of these embodiments, chimeric polypeptide-expressing cells are produced by introducing into them DNA or RNA encoding the chimeric polypeptide by one of a variety of methods, including transduction with a viral vector or transfection of DNA or RNA.

The cell proposed in the present invention may be an ex vivo cell of the subject. The cell may be derived from a sample of peripheral blood mononuclear cells (PBMCs). The cells may be activated and/or expanded prior to being transduced with a nucleic acid encoding molecules enabling the synthesis of a chimeric polypeptide according to the first aspect of the present invention, for example, by being exposed to an anti-CD3 monoclonal antibody.

The cell proposed in the present invention can be obtained by:

(i) isolating a sample containing cells from the subject or from other sources listed above; and

(ii) transducing or transfecting cells with one or more nucleic acid sequences encoding the chimeric polypeptide.

The cells can then be purified, for example, by selection for expression of the antigen binding domain of the antigen binding polypeptide.

PHARMACEUTICAL COMPOSITION

The present invention also relates to a pharmaceutical composition containing a plurality of cells according to the present invention.

The pharmaceutical composition may further contain a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally contain one or more additional pharmaceutically active polypeptides and/or compounds. Such a composition may, for example, be in a form suitable for intravenous infusion.

TREATMENT METHOD

The present invention provides a method of treating a disease, which includes the step of administering to a subject the cells provided by the present invention (eg, in a pharmaceutical composition as described above).

A method for treating a disease relates to the therapeutic use of cells provided in the present invention. According to the present application, cells can be administered to a subject with a disease or condition to alleviate, mitigate or improve at least one symptom associated with the disease and/or to slow, suppress or stop the progression of the disease.

A method for preventing a disease relates to the prophylactic use of cells proposed in the present invention. According to the present application, cells can be administered to a subject who has not yet developed a disease, and/or who does not yet have any symptoms of a disease, to prevent or eliminate the cause of a disease, or to suppress or prevent the occurrence of at least one symptom associated with a disease. The subject may have a predisposition to the disease, or may be at risk of developing the disease.

The method may comprise the steps:

(i) isolating a sample containing cells;

(ii) transducing or transfecting such cells with the nucleic acid sequence or vector proposed in the present invention;

(iii) administering to the subject cells from (ii).

The cell-containing sample may be isolated from the subject or from other sources as described above.

The present invention provides a cell according to the present invention for use in the treatment and/or prevention of a disease.

The present invention also relates to the use of a cell according to the present invention in the production of a medicament for the treatment of a disease.

The disease to be treated by the methods proposed in the present invention may be an oncological disease such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.

The disease may be multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), neuroblastoma, T-cell acute lymphoblastic leukemia (T-ALL), or diffuse large B-cell lymphoma (DLBCL) .

The disease may be a plasma cell disorder such as plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary plasmacytoma of bone, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, or indolent multiple myeloma.

The cells of the present invention may have the ability to kill target cells, such as cancer cells. The target cell may be characterized by the presence of a tumor-secreted ligand or chemokine ligand in close proximity to the target cell. The target cell may be characterized by the presence of a soluble ligand, simultaneously with the expression of tumor-associated antigen (TAA) on the surface of the target cell.

The cells and pharmaceutical compositions provided by the present invention can be used to treat and/or prevent the diseases described above.

The present invention will be further described by way of Examples, which are intended to assist one of ordinary skill in the art in practicing the present invention and are in no way intended to limit the scope of the present invention.

EXAMPLES

Example 1 - BCMA Expression on the Surface of Multiple Melanoma Cells

Primary myeloma cells were isolated by immunomagnetic selection for CD138 from fresh bone marrow samples from patients with multiple myeloma who had advanced disease. These cells were labeled with the BCMA-specific mAb J6MO (GSK) conjugated to PE. At the same time, a bead-based standard with a known number of binding sites was prepared using a PE Quantibrite bead kit (Becton Dickenson), according to the manufacturer's instructions. The copy number of BCMA on myeloma cells was determined by comparing the mean fluorescence intensity of myeloma cells to the standard curve obtained from the bead assay. The range of BCMA copy numbers on the melanoma cell surface was found to be low: 348.7–4268.4 BCMA copies per cell, with a mean of 1181 and a median of 1084.9 (Figure 1). This is significantly lower than, for example, CD19 and GD2, which are classical CAR targets.

Example 2 - Construction of Anti-BCMA FabCAR and Comparison with Anti-BCMA scFvCAR

A panel of CAR-encoding nucleic acid constructs was created as follows:

RQR8-2A-aBCMAscFv-CD8STK-41BBZ - CAR design based on single-chain Fv

RQR8-2A-aBCMAFAb-CD8STK-41BBZ - Fab-based CAR design

RQR8-2A-aBCMA_C11D5.3-CD8STK-41BB-z - bb2121, scFv CAR construct (positive control)

The anti-BCMA scFv and Fab domains contained the VH and VL sequences for the anti-BCMA AT1 described above (SEQ ID NO: 5 and 6, respectively). All constructs were expressed simultaneously with the cell suicide marker gene RQR8, which is described in WO2013/153391, with BCMA binding fragments in both scFv and Fab formats.

Example 3 - Testing CAR Expression

To compare CAR expression in different formats, T cells were transduced with scFv or FabCAR, and the presence of receptors on the cell surface was assessed by flow cytometry. CAR expression was determined by simultaneously staining T cells with soluble BCMA-FC and QBEND10 (to detect RQR8 expression).

Example 4 - FACS Based Cytotoxicity Test (FBK)

The ability of CAR T cells to kill target cells expressing BCMA was assessed using a FACS-based cytotoxicity test. T cells were cultured with target cells in ratios (effector:target) of 1:4 and 1:8. SupT1 cells were used as target cells, which, as a result of modification, expressed a physiological level of BCMA: an average of 686 copies of BCMA per cell (SupT1-BCMA low). Target cells were also generated that expressed very low levels of BCMA: an average of 81 copies of BCMA per cell (JeKo-1). Unmodified SupT1 cells (SupT1-NT) were used as a negative control. Tests were carried out in a 96-well plate in a total volume of 0.2 ml; 5x104 transduced cells were added to each well. Co-cultures were formed after normalization for transduction efficiency. FBK results were assessed after 24 and 72 hours of incubation.

The results are presented in Figures 5 and 6. After 24 hours of co-culture (Figure 5), BCMA scFvCAR and BCMA Fab CAR demonstrated successful killing of target cells expressing low levels of BCMA antigen. However, BCMA FabCAR demonstrated higher efficacy than the equivalent scFvCAR in killing target cells expressing very low levels of BCMA antigen (JeKo-1). BCMA FabCAR also demonstrated more effective killing of target cells compared to another BCMA scFvCAR, bb2121. After 72 hours (Figure 6), a similar pattern was observed. At a 1:8 (E:M) ratio, BCMA FabCAR achieved almost complete killing of target cells with very low BCMA expression, whereas scFvCAR with an equivalent binding element showed only partial cell killing.

Example 5 - Cytokine Release

Secretion of IL-2 and interferon-gamma (IFN-γ) by CAR T cells was assessed by collecting supernatant from co-cultures after 24 hours of culture as described in Example 4 at effector:target ratios of 1:4 and 1:8. The production of IL-2 and IFN-γ was determined by ELISA.

The results for IFN-γ in Figure 7 are shown for co-cultures with target cells without BCMA antigen expression (Figure 7A), and with target cells expressing low levels of BCMA (Figure 7B). BCMA FabCAR-expressing T cells, when co-cultured with low BCMA-expressing target cells, produced levels of IFN-γ comparable to those produced by equivalent scFv CAR T cells. With untransduced SupT1 target cells, a higher background was observed for bb2121 and the equivalent scFvCAR than for FabCAR.

Example 6 - Proliferation Assay

To assess proliferation, the same panel of CAR-expressing T cells described in Example 4 was labeled with Cell Trace Violet (CTV). T cells were incubated with CTV for 20 minutes at 37oC. After this, the reaction was stopped by adding 5 volumes of complete medium. After 5 min of incubation, T cells were washed and resuspended in 2 ml of complete medium. An additional 10 min incubation at room temperature was performed to ensure hydrolysis of the acetate and retention of the dye. Labeled T cells were co-cultured with target cells without BCMA expression (SupT1 NT), with low BCMA expression (SupT1BCMAlow), with very low BCMA expression (JeKo-1) or with MM.1s target cells in a 1:1 ratio within 96 hours. The test was carried out in a 96-well plate in a total volume of 0.2 ml; 5x104 transduced T cells and the same number of target cells were added to each well (1:1 ratio). After coculture, T cells were analyzed by flow cytometry to measure the degree of CTV dilution that occurs when T cells divide.

Cells were also gated for CD3 and the RQR8 marker, then CD8+ or CD8− to determine the number of whole CD4 and CD8 T cells. The results are presented in Figure 8. After culture with all three BCMA-expressing target cells (low expression, very low expression, and MM.1s), cultures with BCMA FabCAR showed more CD8 cells compared to cultures with equivalent scFv CAR and bb2121, reflecting greater proliferation and/or lower cell death (Figure 8B). It should be noted that the background proliferation of CD4 cells with bb2121 and scFvCAR was higher than with Fab CAR. This may provide greater apparent proliferation of target cells, but actually reflects increased CAR activation in the absence of antigen.

Example 7 - Design and Construction of Antibodies with Dual Variable Domains Equivalent to the Antigen Binding Domain of Cleaved Fab CAR

The cleavable Fab CAR is schematically depicted in Figure 9. It contains two antigen binding domains: outer (membrane distal) binding domain 1 and inner (membrane proximal) binding domain 2. Binding domains 1 and 2 are connected by a cleavable linker. Cleavage of the linker, for example by matrix metalloproteinase (MMP), eliminates binding domain 1 from the CAR. Without cleavage, with FabCAR intact, the internal binding domain is partially occluded by the presence of the external domain due to steric hindrance. The internal domain is activated after cleavage of the heavy and light chains.

To demonstrate the possibility of designing a molecule with two binding domains and activating the internal binding domain by cleaving the external binding domain with an enzyme, a soluble antibody was developed as shown in Figure 10.

The antibody contained a first antigen binding domain that binds BCMA and has the VH and VL sequences shown above as SEQ ID NO: 29 and 30, respectively. The second antigen binding domain binds CD19 and has the VH and VL sequences identified above as SEQ ID NO: 67 and 68, respectively.

An MMP-9 cleavable linker having the sequence PLGLAG (SEQ ID NO: 87) was added between the two VL domains and between the two VH domains. A randomized version of this sequence was used as a negative control: LALGPG (SEQ ID NO: 88).

The structures were composed as follows:

Plasmid 1: D8_MMP9_CAT19_HC_HuIgG1

Plasmid 2: D8_scrambled_CAT19_HC_HuIgG1

Plasmid 3: D8_MMP9_CAT19_LC

Plasmid 4: D8_scrambled_CAT19_LC

Chinese hamster ovary (CHO) cells were simultaneously transfected with Plasmid 1 and Plasmid 3, resulting in the antibody shown in Figure 10 with a cleavable MMP linker; or were simultaneously transfected with Plasmid 2 and Plasmid 4, causing them to produce an equivalent antibody with a randomized linker on the VH-containing chain and on the VL-containing chain.

Example 8 - Demonstration of Enzymatic Cleavage of the External Binding Domain of an Antibody Having Two Binding Domains

The antibody described in Example 7 contained an MMP-9 cleavable linker between the VH domains and the VL domains of the BCMA binding domain and the CD19 binding domain. To demonstrate the ability of the BCMA binding domain to be cleavable by the MMP-9 enzyme, a cleavage test was performed as described below.

The concentration of human MMP-9 in the assay buffer was adjusted to 100 μg/ml. To activate the enzyme, P-aminophenylacetate mercury (APMA) was added at a final concentration of 1 mM and incubated at 37°C for 24 hours.

To perform cleavage tests, 50 μg of antibody and 6 μl of activated enzyme were mixed in a final volume of 200 μl buffer. Mixtures for digestion tests were incubated at 37°C and 1 μl of antibody solution (4 μl) was withdrawn at different time points, the collected samples were resuspended in NuPAGE loading dye, boiled, frozen, and then subjected to dodecyl sulfate-polyacrylamide gel electrophoresis sodium (DNS-PAGE). The percentage of cleavage was assessed by densitometry, the results are presented in Figure 11.

The antibody containing the MMP-9 cleavable linker was degraded by matrix metalloproteinase over time, reaching a plateau after approximately 1 hour of exposure to the enzyme. The randomized linker antibody did not show a significant increase in cleavage even after 24 hours of enzyme exposure.

Example 9 - Study of the effect of cleavage of the external binding domain on the binding of a target antigen by the internal binding domain

To study the activity of the internal binding domain with and without the removal of the external binding domain, CD19 binding was assessed by ELISA. Recombinant CD19 was applied to the bottom of the wells of a 96-well plate at a concentration of 1 μg/ml, 50 μl/well. After blocking, antibodies were added to the wells for 30 minutes at a concentration of 15 μl/ml, as described in Example 7 (containing an MMP-9-cleavable linker or a random linker, and previously exposed and not exposed to MMP-9), then washed phosphate-buffered saline (PBS) containing 0.05% Tween 20. Secondary anti-human Fc antibody conjugated to horseradish peroxidase (HRP) was added at a dilution of 1:3000 and incubated for 1 hour. The signal was recorded using 1-Step Ultra TMB substrate and blocked with 1 M H2SO4 solution. The results are presented in Figure 12. The soluble antibody (D8-MMP-CAT19) showed increased binding to CD19 after MMP-9 cleavage, whereas pre-exposure to MMP-9 had no effect on the random linker antibody (D8-scrambled-CAT19) binding to CD19.

Example 10 - Study of the effect of cleavage of the external binding domain on CD19 binding kinetics

CD19 binding kinetics were analyzed using the Biacore 8K biosensor by detecting antibodies on a Protein A-coated chip. The antibody described in Example 8, with or without prior exposure to MMP-9, and a positive control antibody (CAT19 IgG) recorded at 75 resonance units (RU) for IgG and at 100 RU for constructs with two binding domains. Recombinant CD19 was dialyzed in HBS-EP+ buffer, and the analyte was analyzed at a concentration of 500 gM with serial 2-fold dilution. The results are presented in Figure 13. The antibody with the MMP-9 cleavable linker exhibited a higher maximum observed level of binding (Rmax) of CD19 after MMP-9 cleavage (D8-MMP9-CAT19 cleaved) than the equivalent antibody not exposed to MMP-9 (D8 -MMP9-CAT19).

All publications mentioned in the description above are incorporated herein by reference. Various modifications and variations of the described methods and systems proposed in the present invention will become apparent to those skilled in the art within the scope and spirit of the invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as described herein should not be unduly limited to such specific embodiments. Indeed, it is intended that various modifications of the described methods of implementing the present invention, understandable to those skilled in molecular biology and related fields, are within the scope of the following claims.

--->

LIST OF SEQUENCES

<110> Autolus Limited

<120> Chimeric antigen receptor

<130> P116016PCT

<150> GB 1815775.0

<151> September 27, 2018

<150> GB 1902021.3

<151> February 14, 2019

<160> 88

<170> PatentIn version 3.5

<210> 1

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> kappa chain constant domain

<400> 1

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 2

<211> 97

<212> PROTEIN

<213> Artificial sequence

<220>

<223> sequence from immunoglobulin gamma heavy chain

<400> 2

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

1 5 10 15

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

20 25 30

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

35 40 45

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

50 55 60

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

65 70 75 80

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg

85 90 95

Val

<210> 3

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> spacer, human Ig G1 hinge region

<400> 3

Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

Lys Asp Pro Lys

20

<210> 4

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> spacer, articulated spacer

<400> 4

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 5

<211> 124

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH (variable heavy chain region) AT1 anti-BCMA

(maturation antigen

B cells)

<400> 5

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Tyr Met Ser Trp Leu Lys Gln Val Pro Gly Gln Ser Ile Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Ala Gly Asp Gly Ala Thr His Tyr His Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Leu Tyr Phe Cys

85 90 95

Ala Arg Pro Leu Tyr Thr Thr Ala Tyr Tyr Tyr Val Gly Gly Phe Ala

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 6

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL (variable region light chain) AT1 anti-BCMA

<400> 6

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Thr Val Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ser Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Gln Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Ile Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Asp Thr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 7

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT2 vs BCMA

<400> 7

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Ser Gly Asp Thr Ile Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Lys Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Asp Tyr Tyr Asp Gly Tyr Gln Ser Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT2 vs BCMA

<400> 8

Asn Thr Val Met Thr Gln Ser Pro Thr Ser Met Ser Ile Ser Val Gly

1 5 10 15

Asp Arg Val Thr Met Asn Cys Lys Ala Ser Gln Asn Val Gly Asn Asn

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Gln Ala

65 70 75 80

Glu Asp Ala Ala Phe Tyr Tyr Cys Gln Arg Ile Tyr Asn Ser Ala Leu

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT3 vs BCMA

<400> 9

Gln Val Gln Leu Gln Gln Ser Gly Ala Ala Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Val Ser Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Asn Ser Gly Pro Thr Asn Phe Asn Lys Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Thr Pro Arg Thr Val Ala Pro Tyr Asn Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 10

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT3 vs BCMA

<400> 10

Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly Glu

1 5 10 15

Arg Val Ser Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Thr Arg Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile Tyr

35 40 45

Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp

65 70 75 80

Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Trp Asn Asp Pro Tyr Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 11

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT4 vs BCMA

<400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Ile Phe Ser Asn Phe

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Arg Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Thr Thr Ser Gly Gly Asp Thr His Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg His Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Val Tyr Tyr Gly Leu Phe Trp Phe Phe Asp Phe Trp Gly

100 105 110

Pro Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 12

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT4 vs BCMA

<400> 12

Asn Thr Val Met Thr Gln Ser Pro Lys Ser Ile Phe Ile Ser Val Gly

1 5 10 15

Asp Arg Val Thr Val Asn Cys Lys Ala Ser Gln Asn Val Gly Thr Asn

20 25 30

Val Asp Trp Tyr Gln Gln Lys Thr Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser Asn Thr Asn Pro Phe

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 13

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT5 vs BCMA

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Ser Gly Asp Thr Ile Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Lys Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Asp Tyr Tyr Asp Gly Tyr Gln Ser Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT5 vs BCMA

<400> 14

Asp Ile Val Met Thr Gln Ser Pro Ser Thr Leu Pro Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Pro Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln Asp Ala Ser Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 15

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT6 vs BCMA

<400> 15

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Pro Ile Thr Asn Asn

20 25 30

Tyr Asp Trp Ser Trp Ile Arg Gln Phe Pro Gly Asn Lys Met Glu Trp

35 40 45

Met Gly Tyr Ile Ser Asp Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gly Tyr Ile Ser Tyr Ile Pro Phe Ala Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 16

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT6 vs BCMA

<400> 16

Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Leu Gly Gln

1 5 10 15

Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Ile Ser Ser

20 25 30

Tyr Asn Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys

35 40 45

Leu Leu Ile Tyr Asp Ala Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg

50 55 60

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro

65 70 75 80

Val Gln Ala Asp Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ser Lys Asp

85 90 95

Asp Pro Asn Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 17

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT7 vs BCMA

<400> 17

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Pro Ile Thr Asn Asn

20 25 30

Tyr Asp Trp Ser Trp Ile Arg Lys Phe Pro Gly Asn Lys Met Glu Trp

35 40 45

Met Gly Tyr Ile Ser Asp Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Ser Gly Tyr Ile Ser Tyr Ile Pro Phe Gly Phe Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 18

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT7 vs BCMA

<400> 18

Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly Glu

1 5 10 15

Arg Val Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Thr Arg Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile Tyr

35 40 45

Gly Ala Ser Asn Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp

65 70 75 80

Asp Thr Ala Thr Tyr Phe Cys Gln Gln Ser Trp Asn Asp Pro Pro Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 19

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT8 vs BCMA

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Ser Gly Asp Thr Ile Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Lys Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Asp Tyr Tyr Asp Gly Tyr Gln Ser Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT8 vs BCMA

<400> 20

Asp Ile Val Met Thr Gln Ser Pro Ala Ser Gln Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Gly Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Tyr Cys Gln Gln

85 90 95

His Tyr Ser Tyr Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 21

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT9 vs BCMA

<400> 21

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Ser Gly Asp Thr Ile Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Arg His Gly Tyr Tyr Asp Gly Tyr Gln Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 22

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT9 vs BCMA

<400> 22

Asn Thr Val Met Thr Gln Ser Pro Lys Ser Met Ser Ile Ser Val Gly

1 5 10 15

Asp Arg Val Thr Met Asn Cys Lys Ala Ser Gln Asn Val Gly Asn Asn

20 25 30

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Val Gln Ala

65 70 75 80

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Tyr Pro Ser Ile

85 90 95

Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 23

<211> 122

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT10 vs BCMA

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Thr Arg Gly Asp Tyr Gly Tyr Asn Tyr Ala Tyr Trp Phe Ala Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 24

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT10 vs BCMA

<400> 24

Asp Ile Val Met Thr Gln Ala Pro Ser Ser Met Pro Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Pro Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln Tyr Asp Ser Ser Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 25

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT11 vs BCMA

<400> 25

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Thr Ser Gly Asp Ser Ile Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Val Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg His Gly Tyr Tyr Asp Gly Tyr Gln Ser Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 26

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT12 vs BCMA

<400> 26

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Gly Ile Ser Asn Asn

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Ile Lys Pro Leu Ile

35 40 45

Tyr Tyr Thr Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Asp Glu Thr Phe Pro Tyr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 27

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT13 vs BCMA

<400> 27

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Asp Met Ala Trp Val Arg Gln Ala Pro Thr Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Pro Ser Gly Gly Ser Thr Tyr Tyr Arg Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Thr His Asn Tyr Tyr Asp Gly Ser Ser Leu Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 28

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT13 vs BCMA

<400> 28

Asp Ile Val Leu Thr Gln Ser Pro Ala Leu Ala Val Ser Pro Gly Glu

1 5 10 15

Arg Val Thr Ile Ser Cys Gly Ala Asn Glu Thr Val Ser Thr Leu Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Gln Pro Lys Leu Leu Ile Tyr

35 40 45

Leu Ala Ser His Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Pro Val Glu Ala Asp

65 70 75 80

Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Ser Trp Asn Asp Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 29

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH AT14 vs BCMA

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Asp Tyr

20 25 30

Asn Met Ala Trp Val Arg Gln Ala Pro Lys Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ile Tyr Asp Gly Ser Ser Thr Asn His Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Tyr

65 70 75 80

Leu Gln Met Asp Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Thr Arg Pro Gly Pro Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser

115

<210> 30

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL AT15 vs BCMA

<400> 30

Asp Val Val Leu Thr Gln Thr Pro Pro Thr Leu Ser Ala Thr Ile Gly

1 5 10 15

Gln Ser Val Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Phe Leu Ile Tyr Leu Val Ser Gly Leu Gly Ser Gly Val Pro

50 55 60

Asn Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Val His Gly

85 90 95

Thr His Ala Trp Thr Val Gly Gly Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 31

<211> 247

<212> PROTEIN

<213> Artificial sequence

<220>

<223> transmembrane activator and calcium modulator partner and

cyclophilin ligand (CAML) (TACI), 2H6 ScFv

<400> 31

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Arg Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Asn Asp Asp Thr Lys Tyr Thr Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr His Gly Asp Tyr Tyr Ala Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ala Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala

130 135 140

Ser Leu Ala Val Ser Leu Gly Gln Ser Val Thr Ile Ser Cys Arg Ala

145 150 155 160

Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Ala Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn

180 185 190

Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Ser Leu Asn Ile His Pro Val Glu Glu Asp Asp Ile Ala Met

210 215 220

Tyr Phe Cys Gln Gln Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Arg

245

<210> 32

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2H6 binding domain, CDR H1

<400> 32

Asn Tyr Val Met His

15

<210> 33

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2H6 binding domain, CDR H2

<400> 33

Tyr Ile Asn Pro Ser Asn Asp Asp Thr Lys Tyr Thr Glu Lys Phe Lys

1 5 10 15

Gly

<210> 34

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2H6 binding domain, CDR H3

<400> 34

Gly Thr His Gly Asp Tyr Tyr Ala Leu Asp Tyr

1 5 10

<210> 35

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2H6 binding domain, CDR L1

<400> 35

Arg Ala Ser Glu Ser Val Glu Tyr Tyr Gly Thr Ser Leu Met Gln

1 5 10 15

<210> 36

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2H6 binding domain, CDR L2

<400> 36

Gly Ala Ser Asn Val Glu Ser

15

<210> 37

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2H6 binding domain, CDR L3

<400> 37

Gln Gln Ser Arg Lys Val Pro

15

<210> 38

<211> 245

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain, 2G2 ScFv

<400> 38

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Met Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Ala Gln Tyr Ser Asn Pro Ala

50 55 60

Leu Arg Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Asn Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ser Arg Ile His Ser Tyr Tyr Ser Tyr Asp Glu Gly Phe Ala Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln

130 135 140

Ser Gln Lys Phe Met Ser Thr Thr Val Gly Asp Arg Val Ser Ile Thr

145 150 155 160

Cys Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala Trp Tyr Gln Gln

165 170 175

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg

180 185 190

Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp

195 200 205

Phe Thr Leu Thr Ile Ser Asn Met Gln Ser Glu Asp Leu Ala Asp Tyr

210 215 220

Phe Cys Gln Gln Tyr Ser Ser Tyr Arg Thr Phe Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 39

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2G2 binding domain, CDR H1

<400> 39

Thr Phe Gly Met Gly Val Gly

15

<210> 40

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2G2 binding domain, CDR H2

<400> 40

His Ile Trp Trp Asp Asp Ala Gln Tyr Ser Asn Pro Ala Leu Arg Ser

1 5 10 15

<210> 41

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2G2 binding domain, CDR H3

<400> 41

Arg Ile His Ser Tyr Tyr Ser Tyr Asp Glu Gly Phe Ala Tyr

1 5 10

<210> 42

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2G2 binding domain, CDR L1

<400> 42

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 43

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2G2 binding domain, CDR L2

<400> 43

Ser Ala Ser Asn Arg Tyr Thr

15

<210> 44

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 2G2 binding domain, CDR L3

<400> 44

Gln Gln Tyr Ser Ser Tyr

15

<210> 45

<211> 115

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain, 1G6 VH

<400> 45

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Gly Val Asp Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Ile Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser Ala Phe Lys

50 55 60

Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Ser Gly Asp Arg Ala Ala Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

100 105 110

Val Ser Ser

115

<210> 46

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain, 1G6 VL

<400> 46

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Thr Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Val Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn Met Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 47

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain 1G6, CDR H1

<400> 47

Ser Tyr Gly Val Asp

15

<210> 48

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 1G6 binding domain, CDR H2

<400> 48

Ile Ile Trp Gly Gly Gly Arg Thr Asn Tyr Asn Ser Ala Phe Lys Ser

1 5 10 15

<210> 49

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain 1G6, CDR H3

<400> 49

Gly Asp Arg Ala Ala Asp Tyr

15

<210> 50

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 1G6 binding domain, CDR L1

<400> 50

Lys Ala Ser Gln Asn Val Gly Thr Ala Val Ala

1 5 10

<210> 51

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 1G6 binding domain, CDR L2

<400> 51

Ser Ala Ser Asn Arg Tyr Thr

15

<210> 52

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 1G6 binding domain, CDR L3

<400> 52

Gln Gln Tyr Ser Ser Tyr Pro

15

<210> 53

<211> 114

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain, 4B11 VH

<400> 53

Glu Val Gln Leu Gln Gln Ser Val Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asn Thr

20 25 30

Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Lys Ile Asp Pro Ala Asn Gly Asn Ser Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Thr Ile Tyr Tyr Cys

85 90 95

Thr Ser Gly Tyr Gly Ala Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val

100 105 110

Ser Ser

<210> 54

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain, 4B11 VL

<400> 54

Asp Ile Val Leu Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ile Gly

1 5 10 15

Glu Lys Val Thr Leu Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Phe Gln Gln Lys Pro Gly Gln

35 40 45

Ser Leu Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Phe Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Thr Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Thr Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 55

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 4B11 binding domain, CDR H1

<400> 55

Asn Thr Tyr Ile His

15

<210> 56

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 4B11 binding domain, CDR H2

<400> 56

Lys Ile Asp Pro Ala Asn Gly Asn Ser Glu Tyr Ala Pro Lys Phe Gln

1 5 10 15

Gly

<210> 57

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 4B11 binding domain, CDR H3

<400> 57

Gly Tyr Gly Ala Tyr

15

<210> 58

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI binding domain 4B11, CDR L1

<400> 58

Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 59

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 4B11 binding domain, CDR L2

<400> 59

Trp Ala Ser Thr Arg Glu Phe

15

<210> 60

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI 4B11 binding domain, CDR L3

<400> 60

Gln Gln Tyr Tyr Thr Trp

15

<210> 61

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR VH CD19-binding domain, CDR1

<400> 61

Gly Tyr Ala Phe Ser Ser Ser

15

<210> 62

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR VH CD19-binding domain, CDR2

<400> 62

Tyr Pro Gly Asp Glu Asp

15

<210> 63

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR VH CD19-binding domain, CDR3

<400> 63

Ser Leu Leu Tyr Gly Asp Tyr Leu Asp Tyr

1 5 10

<210> 64

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR VL CD19-binding domain, CDR1

<400> 64

Ser Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 65

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR VL CD19-binding domain, CDR2

<400> 65

Asp Thr Ser Lys Leu Ala Ser

15

<210> 66

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDR VL CD19-binding domain, CDR3

<400> 66

Gln Gln Trp Asn Ile Asn Pro Leu Thr

15

<210> 67

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH sequence of mouse monoclonal antibody

<400> 67

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Glu Asp Thr Asn Tyr Ser Gly Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Leu Leu Tyr Gly Asp Tyr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 68

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH CDR1

<400> 68

Gly Phe Thr Phe Ser Asp Ser Tyr

15

<210> 69

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH CDR2

<400> 69

Ile Tyr Ala Gly Asp Gly Ala Thr

15

<210> 70

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH CDR3

<400> 70

Ala Arg Pro Leu Tyr Thr Thr Ala Tyr Tyr Tyr Val Gly Gly Phe Ala

1 5 10 15

Tyr

<210> 71

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL CDR1

<400> 71

Gln Ser Leu Leu Ser Ser Gly Asn Gln Lys Asn Tyr

1 5 10

<210> 72

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL CDR2

<400> 72

Trp Ala Ser

1

<210> 73

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL CDR3

<400> 73

Gln Gln Tyr Tyr Asp Thr Pro Leu Thr

15

<210> 74

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> TACI cysteine-rich domain (CRD) DXL motif

<220>

<221> OTHER_CHARACTERISTIC

<222> (1)..(1)

<223> Xaa may be Phe, Tyr or Trp

<220>

<221> other_sign

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> OTHER_CHARACTERISTIC

<222> (5)..(5)

<223> Xaa can be Val or Thr

<220>

<221> OTHER_CHARACTERISTIC

<222> (6)..(6)

<223> Xaa can be Arg or Gly

<400> 74

Xaa Asp Xaa Leu Xaa Xaa

15

<210> 75

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL sequence of mouse monoclonal antibody

<400> 75

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Phe Leu Thr Ile Asn Asn Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ile Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg

100 105

<210> 76

<211> 241

<212> PROTEIN

<213> Artificial sequence

<220>

<223> mouse monoclonal antibody scFv sequence

<400> 76

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Tyr Pro Gly Asp Glu Asp Thr Asn Tyr Ser Gly Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Leu Leu Tyr Gly Asp Tyr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro

180 185 190

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Phe Leu Thr Ile

195 200 205

Asn Asn Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Asn Ile Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Arg

<210> 77

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> cleavage site, 2A-like sequence

<400> 77

Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu

1 5 10 15

Asn Pro Gly Pro

20

<210> 78

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> glycine-serine linker

<400> 78

Gly Gly Gly Gly Ser

15

<210> 79

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH CDR1

<400> 79

Gly Phe Ile Phe Ser Asp Tyr Asn

15

<210> 80

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH CDR2

<400> 80

Ile Ile Tyr Asp Gly Ser Ser Thr

15

<210> 81

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VH CDR3

<400> 81

Ala Thr Arg Pro Gly Pro Phe Ala Tyr

15

<210> 82

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL CDR1

<400> 82

Gln Ser Leu Leu His Ser Asn Gly Asn Thr Tyr

1 5 10

<210> 83

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL CDR2

<400> 83

Leu Val Ser

1

<210> 84

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> VL CDR3

<400> 84

Val His Gly Thr His Ala Trp Thr

15

<210> 85

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MMP-1, MMP cleavable sequence

<400> 85

Pro Leu Gly Leu Trp Ala

15

<210> 86

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MMP-2, MMP cleavable sequence

<400> 86

Pro Ala Gly Leu Ala Gly

15

<210> 87

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> MMP-9, MMP cleavable sequence

<400> 87

Pro Leu Gly Leu Ala Gly

15

<210> 88

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> random linker cleavable by MMP-9 (negative

control)

<400> 88

Leu Ala Leu Gly Pro Gly

15

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Claims (35)

1. A chimeric antigen receptor (CAR) that binds B cell maturation antigen (BCMA), wherein said CAR comprises a Fab antigen binding domain, wherein said Fab antigen binding domain comprises: a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the following sequences: CDR1 – GFIFSDYN (SEQ ID NO: 79), CDR2 – IIYDGSST (SEQ ID NO: 80), CDR3 – ATRPGPFAY (SEQ ID NO: 81), and b) a light chain variable region (VL) having complementarity determining regions (CDRs) with the following sequences: CDR1 – QSLLHSNGNTY (SEQ ID NO: 82), CDR2 – LVS (SEQ ID NO: 83), CDR3 – VHGTHAWT (SEQ ID NO: 84). 2. CAR according to claim 1, characterized in that said antigen-binding domain Fab contains a VH domain having the sequence SEQ ID NO: 29; and a VL domain having the sequence SEQ ID NO: 30. 3. A nucleic acid expression construct for providing expression of a CAR, which contains a nucleic acid molecule encoding a CAR according to claim 1 or 2. 4. A nucleic acid expression construct for providing expression of a CAR according to claim 1 or 2, which has one of the following general structures: VH-CH-spacer-TM-endo-coexpr-VL-CL; VL-CL-coexpr-VH-CH-spacer-TM-endo; VL-CL-spacer-TM-endo-coexpr-VH-CH or VH-CH-coexpr-VL-CL-spacer-TM-endo; Where: VH is a nucleic acid sequence encoding the heavy chain variable region of the Fab CAR domain according to claim 1 or 2; CH is a nucleic acid sequence encoding a heavy chain constant region; spacer is a nucleic acid encoding a spacer; TM is a nucleic acid sequence encoding a transmembrane domain; endo is a nucleic acid sequence encoding an endodomain; coexpr is a nucleic acid sequence allowing co-expression of the first and second polypeptides; VL is a nucleic acid sequence encoding the light chain variable region of the Fab CAR domain according to claim 1 or 2; And CL is a nucleic acid sequence encoding a light chain constant region. 5. A nucleic acid expression construct for providing expression of a CAR according to claim 1 or 2 and a second CAR, which contains a first nucleic acid molecule encoding a CAR according to claim 1 or 2, and a second nucleic acid molecule encoding a second CAR that has an antigen binding domain , which is a domain antibody (dAb), scFv or Fab. 6. The nucleic acid expression construct according to claim 5, characterized in that said second chimeric antigen receptor binds to one of the following antigens: CD19, FcRL5 and TACI. 7. An expression vector for producing a cell having the ability to kill a target cell expressing BCMA, which contains a nucleic acid expression construct according to any one of claims. 3-6. 8. A cytolytic immune cell having the ability to kill a target cell expressing BCMA that expresses a CAR according to any one of claims. 1, 2, wherein said cell contains a nucleic acid encoding a CAR according to any one of claims. 12. 9. A cytolytic immune cell having the ability to kill a target cell expressing BCMA that expresses the first CAR according to any one of claims. 1, 2 and a second chimeric antigen receptor that has an antigen binding domain that is a domain antibody (dAb), scFv or Fab, wherein said cell contains a nucleic acid encoding a CAR according to any one of claims. 12. 10. The method of obtaining a cell according to claim 8, which includes the step of introducing the expression construct according to claim 3 or 4 into a cytolytic immune cell ex vivo . 11. The method of obtaining a cell according to claim 9, which includes the step of introducing the expression construct according to claim 5 or 6 into a cytolytic immune cell ex vivo . 12. A pharmaceutical composition for the treatment of cancer that expresses BCMA, which contains in an effective amount the cells of claim 8 or 9 together with a pharmaceutically acceptable carrier, diluent or excipient. 13. A method of treating cancer that expresses BCMA, said method comprising the step of administering to a subject the pharmaceutical composition of claim 12. 14. The method according to claim 13, characterized in that said cancer is multiple myeloma.