p95HER2-specific chimeric antigen receptor and its use

Abstract

Providing a chimeric antigen receptor specific to p95HER2 and its use. [Solution] The present invention relates to a chimeric antigen receptor (CAR) capable of targeting p95HER2-expressing cells. The present invention also relates to a single-stranded variable fragment (ScFv), an antigen-binding domain, and an antibody or antibody fragment capable of binding to the p95HER2 antigen. The present invention also relates to a method for diagnosing cancer, and to a pharmaceutical composition for use in a method for preventing or treating cancer comprising the CAR and / or antigen-binding domain or antibody of the present invention. The authors of the present invention have obtained a chimeric antigen receptor (CAR) capable of targeting p95HER2-expressing cells and inducing potent antitumor activity against p95HER2-positive tumors, but without apparent activity against cells expressing normal levels of HER2.

Description

[Technical Field]

[0001] This invention falls within the fields of biotechnology and biomedicine. Specifically, it relates to an antibody specific to the HER2 p95 fragment, a chimeric antigen receptor containing said antibody, and its use in the treatment of cancer. [Background technology]

[0002] Cancer is one of the leading causes of incidence and mortality worldwide. Currently, nearly one in six people die from cancer globally, and the number of new cases is expected to increase by approximately 70% in the next 20 years.

[0003] Currently, many drugs are available for use in the treatment of cancer. However, in many cases, cancer does not respond to anticancer therapy, or its growth and / or metastasis is only slowed. Even when tumors initially respond to anticancer therapy by reducing in size or going into remission, they often develop resistance to the drugs. For these reasons, there is a need for new anticancer agents and drugs that can be used to treat cancers for which there are not yet available treatments, as well as for multidrug-resistant cancers.

[0004] HER2 is a receptor tyrosine kinase that is overexpressed in approximately 25% of breast and gastric cancers. Despite successful anti-HER2 therapies such as the monoclonal antibody trastuzumab or the inhibitor lapatinib, a high percentage (40%) of breast cancer cases eventually progress. Furthermore, cardiotoxicity due to HER2 expression in cardiomyocytes is frequently observed in treated patients. Therefore, there is a clinical need to develop more effective and safer treatments for HER2-driven tumors. HER2-targeted carcinomatous angioplasty (CAR) is also being developed. However, adoptive cell therapy for HER2 is limited by HER2 expression in healthy tissue, which leads to serious side effects.

[0005] p95HER2 is a fragment of HER2 that is expressed only in certain HER2-positive tumors. It has been shown that T cells can be safely oriented to p95HER2 via T cell bispecific antibodies. However, adoptive cell therapies based on p95HER2-specific chimeric antigen receptors (CARs) have not been developed. In fact, previous attempts to generate p95HER2 CARs failed to express them on the T cell surface and were unable to kill cells expressing p95HER2 (Research Disclosure, Database No. 667070). Therefore, there is a need in the art for antitumor therapies that specifically target p95HER2-expressing cells. [Overview of the project] [Means for solving the problem]

[0006] The authors of this invention have obtained a chimeric antigen receptor (CAR) that can target p95HER2-expressing cells and induce potent antitumor activity against p95HER2-positive tumors, but has no apparent activity against cells expressing normal levels of HER2. The CAR was obtained using ScFv from anti-p95HER2 ScFv, which has not previously provided a functional CAR and required humanization of ScFv and modification of the VH and VL regions within ScFv to a specific arrangement. This is shown in Example 1 of this document, which demonstrates that the CAR of this invention induces a specific cytotoxic effect on cells expressing p95HER2, and in contrast, has no effect on cells that do not express p95HER2.

[0007] In addition, the authors of this invention have generated CARs from different anti-p95HER2 ScFvs and shown that CARs can induce potent cytotoxic effects on p95HER2-expressing cells. This is shown in Examples 2 and 3 of this document. Furthermore, the use of humanized ScFv versions generates CAR Ts that are more specific to p95HER2 compared to non-humanized versions, due to reduced killing of cells expressing normal levels of HER2, as shown in Figures 6 and 8. Thus, in a first aspect, the present invention is (i) A p95HER2-specific antigen-binding domain, (ii) Transmembrane domain and (iii) With respect to a chimeric antigen receptor (CAR) comprising at least one intracellular signaling domain and / or a costimulatory domain, The antigen-binding domain is, (i) -The framework areas of the VL and VH domains have been humanized, - The VH region contains the sequences of sequence numbers 1, 2, and 3 or their functional equivalent variants, or the sequences of sequence numbers 1, 174, and 3 or their functional equivalent variants, -ScFv(ScFv1) is characterized in that CDR1, CDR2, and CDR3 in the VL region each contain the sequences of SEQ ID NOs. 4, 5, and 6 or their functional equivalent variants, or the sequences of SEQ ID NOs. 175, 5, and 6 or their functional equivalent variants. (ii) - Having at least one VH and at least one VL region, -At least CDR1, CDR2, and CDR3 in the VH region contain the sequences of sequence numbers 7, 8, and 9, or their functionally equivalent variants, -An antigen-binding domain (antigen-binding domain 1) characterized in that at least the VL region CDR1, CDR2, and CDR3 each contain the sequence of SEQ ID NOs. 10, 11, and 12 or a functionally equivalent variant thereof, and (iii) - Having at least one VH and at least one VL region, -At least CDR1, CDR2, and CDR3 in the VH region contain the sequences of sequence numbers 13, 14, and 15, or their functionally equivalent variants, - At least the CDR1, CDR2, and CDR3 of the VL region each contain the sequence of SEQ ID NO: 16, 17, and 18 or a functionally equivalent variant thereof, or the sequence of SEQ ID NO: 179, 17, and 18 or a functionally equivalent variant thereof, and is selected from the group consisting of antigen-binding domains (antigen-binding domain 2).

[0008] In a second aspect, the present invention relates to a nucleic acid encoding the CAR of the present invention.

[0009] In a third aspect, the present invention relates to an expression vector containing the nucleic acid of the second aspect of the present invention.

[0010] In a fourth aspect, the present invention relates to a host cell containing the nucleic acid of the second aspect of the present invention or the vector of the third aspect of the present invention.

[0011] In a fifth aspect, the present invention - The CDR1, CDR2, and CDR3 of the VH region each contain the sequence of SEQ ID NO: 1, 2, and 3 or a functionally equivalent variant thereof, or the sequence of SEQ ID NO: 1, 174, and 3 or a functionally equivalent variant thereof, - The CDR1, CDR2, and CDR3 of the VL region each contain the sequence of SEQ ID NO: 4, 5, and 6 or a functionally equivalent variant thereof, or the sequence of SEQ ID NO: 175, 5, and 6 or a functionally equivalent variant thereof, and relates to a ScFv.

[0012] In a sixth aspect, the present invention - Has at least one VH region and at least one VL region, - The CDR1, CDR2, and CDR3 of at least the VH region each contain the sequence of SEQ ID NO: 7, 8, and 9 or a functionally equivalent variant thereof, - The CDR1, CDR2, and CDR3 of at least the VL region each contain the sequence of SEQ ID NO: 10, 11, and 12 or a functionally equivalent variant thereof, and relates to an antigen-binding domain.

[0013] In a seventh aspect, the present invention relates to having at least one VH region and at least one VL region, - wherein CDR1, CDR2, and CDR3 of at least the VH region each contain the sequences of SEQ ID NOs: 13, 14, and 15 or functional equivalent variants thereof, - wherein CDR1, CDR2, and CDR3 of at least the VL region each contain the sequences of SEQ ID NOs: 16, 17, and 18 or functional equivalent variants thereof, or the sequences of SEQ ID NOs: 179, 17, and 18 or functional equivalent variants thereof, and relates to an antibody or a fragment thereof.

[0014] In one aspect, the present invention relates to a nucleic acid encoding a ScFv, an antigen-binding domain, or an antibody according to the fifth, sixth, and seventh aspects of the present invention.

[0015] In a ninth aspect, the present invention relates to an expression vector containing the nucleic acid of the eighth aspect of the present invention.

[0016] In a tenth aspect, the present invention relates to a host cell containing the nucleic acid of the eighth aspect of the present invention or the expression vector of the ninth aspect of the present invention.

[0017] In an eleventh aspect, the present invention relates to (i) contacting a sample from a patient containing tumor cells with a ScFv, an antigen-binding domain, or an antibody according to the fifth, sixth, or seventh aspect of the present invention, (ii) detecting the binding of the ScFv, antigen-binding domain, or antibody in the sample to the cells, and relates to a method for diagnosing cancer in a patient, The presence of binding indicates that the patient has cancer.

[0018] In a twelfth aspect, the present invention relates to a pharmaceutical composition containing the host cell of the fourth aspect of the present invention and / or a ScFv, an antigen-binding domain, or an antibody according to the fifth, sixth, or seventh aspect of the present invention, and at least one pharmaceutically acceptable excipient and / or vehicle.

[0019] In a thirteenth aspect, the present invention relates to a host cell according to a fourth aspect of the present invention and / or an ScFv, antigen-binding domain, or antibody according to a fifth, sixth, and seventh aspect of the present invention for use in pharmaceuticals.

[0020] In the final aspect, the present invention relates to a host cell according to a fourth aspect of the present invention and / or an ScFv, antigen-binding domain, or antibody according to a fifth, sixth, and seventh aspect of the present invention for use in a method of preventing or treating cancer. In certain embodiments, the following items are provided: (Item 1) It is a chimeric antigen receptor (CAR), (i) A p95HER2-specific antigen-binding domain, (ii) Transmembrane domain and (iii) comprising at least one intracellular signaling domain and / or a co-stimulatory domain, The antigen-binding domain, (i) ScFv(ScFv1), -The framework areas of the VL and VH domains have been humanized, - The VH region CDR1, CDR2, and CDR3 each contain the sequences of sequence numbers 1, 2, and 3 or their functionally equivalent variants, or the sequences of sequence numbers 1, 174, and 3 or their functionally equivalent variants, -ScFv(ScFv1) is characterized in that CDR1, CDR2, and CDR3 in the VL region each contain the sequences of sequence numbers 4, 5, and 6 or their functional equivalent variants, or the sequences of sequence numbers 175, 5, and 6 or their functional equivalent variants. (ii) Antigen-binding domain (antigen-binding domain 1), - Having at least one VH region and at least one VL region, - The CDR1, CDR2, and CDR3 of the at least one VH region are, , including the sequence of sequence numbers 7, 8, and 9 or a functionally equivalent variant thereof, -An antigen-binding domain (antigen-binding domain 1) characterized in that at least one VL region CDR1, CDR2, and CDR3 each contain the sequence of SEQ ID NOs. 10, 11, and 12 or a functionally equivalent variant thereof, (iii) Antigen-binding domain (antigen-binding domain 2), - Having at least one VH region and at least one VL region, - The CDR1, CDR2, and CDR3 of the at least one VH region each contain the sequence of sequence numbers 13, 14, and 15 or a functionally equivalent variant thereof, A chimeric antigen receptor (CAR) selected from the group comprising: an antigen-binding domain (antigen-binding domain 2) characterized in that at least one VL region CDR1, CDR2, and CDR3 each contain the sequence of SEQ ID NOs. 16, 17, and 18 or a functionally equivalent variant thereof, or the sequence of SEQ ID NOs. 179, 17, and 18 or a functionally equivalent variant thereof. (Item 2) The CAR described in item 1, wherein the VL region of ScFv1 is located at the N-terminus with respect to the VH region. (Item 3) The CAR according to item 1 or 2, wherein FR1, FR2, FR2, and FR4 of the VH region of ScFv1 each include the sequences of sequence numbers 19, 20, 21, and 22 or their functionally equivalent variants, and FR1, FR2, FR3, and FR4 of the VL region of ScFv1 each include the sequences of sequence numbers 23, 24, 25, and 26 or their functionally equivalent variants. (Item 4) The CAR described in item 3, wherein VL of ScFv1 includes the sequence of sequence number 27 or 180 or a functionally equivalent variant thereof, and VH includes the sequence of sequence number 28 or 181 or a functionally equivalent variant thereof. (Item 5) The CAR according to any one of items 1 to 3, wherein the VH and VL regions of the ScFv1 are connected by a linker region containing the sequence of sequence number 29. (Item 6) The CAR according to item 5, wherein the ScFv1 comprises the sequence of sequence number 30 or 182 or a functionally equivalent variant thereof. (Item 7) The CAR according to item 1, wherein FR1, FR2, FR3, and FR4 of at least one VH region of the antigen-binding domain 1 each contain the sequence of SEQ ID NOs. 31, 32, 33, and 34, SEQ ID NOs. 65, 66, 67, and 68, or SEQ ID NOs. 73, 74, and 75, or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of the antigen-binding domain 1 each contain the sequence of SEQ ID NOs. 35, 36, 37, and 38, SEQ ID NOs. 69, 70, 71, and 72, or SEQ ID NOs. 77, 78, 79, and 80, or a functionally equivalent variant thereof. (Item 8) The CAR according to item 7, wherein the at least one VL region of the antigen-binding domain 1 comprises the sequence of SEQ ID NO: 39, 54, or 56 or a functionally equivalent variant thereof, and the at least one VH region of the antigen-binding domain 1 comprises the sequence of SEQ ID NO: 40, 53, or 55 or a functionally equivalent variant thereof. (Item 9) The CAR according to item 7 or 8, wherein the antigen-binding domain 1 is ScFv, and the VH and VL regions of the ScFv are connected by a linker region including SEQ ID NO: 29. (Item 10) ScFv is the sequence of sequence numbers 41, 187, 188, or 189 or its functional equivalent CARs as described in item 9, including valence variants. (Item 11) The CAR according to item 1, wherein FR1, FR2, FR3, and FR4 of at least one VH region of the antigen-binding domain 2 each contain the sequence of SEQ ID NOs. 42, 43, 44, and 45, SEQ ID NOs. 89, 90, 91, and 92, or SEQ ID NOs. 97, 98, 99, and 100, or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of the antigen each contain the sequence of SEQ ID NOs. 46, 47, 48, and 49, or SEQ ID NOs. 93, 94, 95, and 96, or a functionally equivalent variant thereof. (Item 12) The CAR according to item 11, wherein the at least one VL region includes the sequence of sequence numbers 50, 184, 60, or 62 or a functionally equivalent variant thereof, and the at least one VH includes the sequences of sequence numbers 51, 59, and 61 or a functionally equivalent variant thereof. (Item 13) The CAR according to item 11 or 12, wherein the antigen-binding domain 2 is an ScFv, and the VH and VL regions of the ScFv are connected by a linker region including sequence number 29. (Item 14) The CAR according to item 13, wherein the ScFv includes the sequence of sequence numbers 52, 186, 190, or 191 or a functionally equivalent variant thereof. (Item 15) A CAR according to any one of items 1 to 14, further comprising a hinge domain between the antigen-binding domain and the transmembrane domain. (Item 16) The CAR described in item 15, wherein the aforementioned hinge domain is the CD8 hinge domain. (Item 17) The CAR described in any one of items 1 to 16, wherein the transmembrane domain is selected from the group consisting of a CD4 transmembrane domain, a CD8 transmembrane domain, a CD28 transmembrane domain, a 4-1BB transmembrane domain, a CTLA4 transmembrane domain, a CD27 transmembrane domain, and a CD3 zeta transmembrane domain. (Item 18) The CAR according to any one of items 1 to 17, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or any combination thereof. (Item 19) The CAR according to item 18, wherein the at least one intracellular signaling domain comprises an intracellular domain of a co-stimulatory molecule selected from OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof. (Item 20) The CAR according to any one of items 1 to 19, wherein the at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain. (Item 21) The CAR according to item 20, wherein at least one intracellular signaling domain is located N-terminal to the CD3 zeta intracellular domain. (Item 22) The CAR described in item 21, wherein the hinge region is a CD8 hinge domain, the transmembrane domain is a CD28 transmembrane domain, and the intracellular signaling domain is a CD28 costimulatory domain. (Item 23) A nucleic acid encoding a CAR as described in any one of items 1 through 22. (Item 24) The nucleic acid according to item 23, wherein the encoded CAR further comprises a leader sequence. (Item 25) The nucleic acid described in item 24, wherein the aforementioned leader sequence is a CD8 leader sequence. (Item 26) An expression vector containing a nucleic acid as described in any one of items 23-25. (Item 27) A host cell containing a chimeric antigen receptor (CAR) as described in any one of items 1 to 22, a nucleic acid as described in any one of items 23 to 25, or an expression vector as described in item 26. (Item 28) The host cells described in item 27, wherein the aforementioned cells are immune cells. (Item 29) The host cell described in item 28, wherein the immune cell is a T cell, an NK cell, or an NKT cell. (Item 30) The host cells described in item 29, wherein the T cells are CD8+ T cells. (Item 31) It is ScFv, - The VH region contains the sequences of sequence numbers 1, 2, and 3 or their functional equivalent variants, or the sequences of sequence numbers 1, 174, and 3 or their functional equivalent variants, -ScFv characterized in that CDR1, CDR2, and CDR3 of the VL region each contain the sequences of SEQ ID NOs. 4, 5, and 6 or their functional equivalent variants, or the sequences of SEQ ID NOs. 175, 5, and 6 or their functional equivalent variants. (Item 32) The ScFv described in item 31, wherein FR1, FR2, FR3, and FR4 of the VH region each contain the sequence of sequence numbers 152, 153, 154, and 155, sequence numbers 19, 20, 21, and 22, or sequence numbers 163, 164, 165, and 166, or their functionally equivalent variants, and FR1, FR2, FR3, and FR4 of the VL region each contain the sequence of sequence numbers 156, 157, 158, and 159, sequence numbers 23, 24, 25, and 26, or sequence numbers 167, 168, 169, or 170, or their functionally equivalent variants. (Item 33) The ScFv described in item 32, wherein VL includes the sequence of sequence number 160, 193, 27, 171, or 180 or a functionally equivalent variant thereof, and VH includes the sequence of sequence number 161, 194, 28, 172, or 181 or a functionally equivalent variant thereof. (Item 34) The ScFv described in item 33, wherein the VH and VL regions of the ScFv1 are connected by a linker region including sequence number 29. (Item 35) The ScFv described in item 334, wherein the ScFv includes the sequence of sequence numbers 162, 195, 30, 173, or 182 or a functionally equivalent variant thereof. (Item 36) It is an antigen-binding domain, - Having at least one VH region and at least one VL region, - The CDR1, CDR2, and CDR3 of the at least one VH region each contain the sequence of sequence numbers 7, 8, and 9 or their functionally equivalent variants, - The CDR1, CDR2, and CDR3 of the at least one VL region are, An antigen-binding domain characterized by containing the sequences of SEQ ID NOs. 10, 11, and 12 or functionally equivalent variants thereof. (Item 37) The antigen-binding domain according to item 36, wherein FR1, FR2, FR3, and FR4 of at least one VH region each contain the sequence of SEQ ID NOs. 31, 32, 33, and 34, SEQ ID NOs. 65, 66, 67, and 68, or SEQ ID NOs. 73, 74, 75, and 76, and FR1, FR2, FR3, and FR4 of at least one VL region each contain the sequence of SEQ ID NOs. 35, 36, 37, and 38, SEQ ID NOs. 69, 70, 71, 72, or SEQ ID NOs. 77, 78, 79, and 80, or a functionally equivalent variant thereof. (Item 38) The antigen-binding domain according to item 37, wherein the at least one VL region comprises the sequences of SEQ ID NOs. 39, 54, and 56 or functionally equivalent variants thereof, and the at least one VH region comprises the sequences of SEQ ID NOs. 40, 53, and 55 or functionally equivalent variants thereof. (Item 39) The antigen-binding domain according to item 38, wherein the antigen-binding domain 1 is ScFv, and the VH and VL regions of the antigen-binding domain are connected by a linker region including sequence number 29. (Item 40) The antigen-binding domain described in item 39, wherein the ScFv comprises the sequence of sequence number 41, 187, 188, or 189 or a functionally equivalent variant thereof. (Item 41) An antibody or a fragment thereof, - At least one VH region and at least one VL region, - The CDR1, CDR2, and CDR3 of the at least one VH region each contain the sequence of sequence numbers 13, 14, and 15 or a functionally equivalent variant thereof, - An antibody or antibody fragment characterized in that the CDR1, CDR2, and CDR3 of at least one VL region each contain the sequence of SEQ ID NOs. 16, 17, and 18 or a functionally equivalent variant thereof, or the sequence of SEQ ID NOs. 179, 17, and 18 or a functionally equivalent variant thereof. (Item 42) The antibody or antibody fragment according to item 41, wherein FR1, FR2, FR3, and FR4 of at least one VH region each contain the sequence of SEQ ID NOs. 42, 43, 44, and 45, SEQ ID NOs. 89, 90, 91, and 92, or SEQ ID NOs. 97, 98, 99, and 100, or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region each contain SEQ ID NOs. 46, 47, 48, and 49, SEQ ID NOs. 93, 94, 95, and 96, or SEQ ID NOs. 101, 102, 103, and 104, or a functionally equivalent variant thereof. (Item 43) The antibody or antibody fragment according to item 42, wherein the at least one VL region comprises the sequence of SEQ ID NO: 50, 184, 60, or 62 or a functionally equivalent variant thereof, and the at least one VH region comprises the sequence of SEQ ID NO: 51, 59, or 61 or a functionally equivalent variant thereof. (Item 44) The antibody or antibody fragment according to item 43, wherein the antibody fragment is ScFv, and the VH and VL regions are connected by a linker region containing SEQ ID NO: 29. (Item 45) The antibody or antibody fragment described in item 44, wherein the ScFv comprises the sequence of SEQ ID NO: 52, 186, 190, or 191 or a functionally equivalent variant thereof. (Item 46) A nucleic acid encoding an ScFv, antigen-binding domain, or antibody as described in any one of items 31-45. (Item 47) An expression vector containing the nucleic acid described in item 46. (Item 48) A host cell containing the nucleic acid described in item 46 or the expression vector described in item 47. (Item 49) A method for diagnosing cancer in patients, (i) Contacting the patient sample containing tumor cells with the ScFv and antigen-binding domain or antibody described in any one of items 31 to 45, (ii) detecting the binding of the ScFv, antigen-binding domain, or antibody in the sample to cells, A method by which the presence of the aforementioned bond indicates that the patient has cancer. (Item 50) The method according to item 49, wherein the ScFv1, antigen-binding domain, or antibody is disposed on a solid support. (Item 51) A pharmaceutical composition comprising a host cell as described in any one of items 27 to 30, an antibody or antibody fragment as described in any one of items 31 to 45, and at least one pharmaceutically acceptable excipient and / or vehicle. (Item 52) Host cells as described in any one of items 27-30, or antibodies or antibody fragments as described in any one of items 31-45, for use in pharmaceuticals. (Item 53) Host cells as described in any one of items 27-30, or antibody fragments as described in any one of items 31-45, for use in methods of preventing or treating cancer. (Item 54) The aforementioned cancer is p95HER2 positive, and is a host cell or antibody fragment for use as described in item 53. [Brief explanation of the drawing]

[0021] [Figure 1] Schematic diagrams of the three p95HER2 CARs disclosed in this document. (A) Humanized 32H2 ​​p95HER2 CAR. (B) 214D8 p95HER2 CAR. (C) 215C2 p95HER2 CAR. [Figure 2] Design, expression, and cytotoxicity of 32H2 ​​p95HER2 CAR T cells. (A) Schematic diagram of a chimeric receptor containing ScFv that binds to full-length HER2 or p95HER2. (B) Surface expression of the indicated CAR on T cells 5 days post-transduction; the percentage of positive CAR T cells from total T cells is shown. (C) MCF10A p95HER2 cells were co-cultured with CAR T cells in the indicated ratio. After 48 hours, viable target cells were evaluated by flow cytometry. UTD: Untransduced T cells; Trast: Trastuzumab-based CAR. [Figure 3] Design and expression of humanized 32H2 ​​p95HER2 CAR T cells. (A) Schematic diagram of a chimeric receptor containing ScFv that binds to full-length HER2 or p95HER2. (B) Surface expression of the CAR shown in A on T cells 5 days post-transduction; percentage of positive CAR T cells from total T cells is shown. UTD: untransduced T cells; Trast: trastuzumab-based CAR. [Figure 4] Design, expression, and cytotoxicity of h32H2 p95HER2 CAR T cells. (A) Schematic diagram of a chimeric receptor containing ScFv that binds to full-length HER2 or p95HER2. (B) Surface expression of the indicated CAR on T cells 5 days post-transduction; the percentage of positive CAR T cells from total T cells is shown. (C) MCF10A p95HER2 cells were co-cultured with CAR T cells in the indicated ratio. Surviving target cells were evaluated by flow cytometry at 48 hours. (D) MCF10A cells were co-cultured with CAR T cells in the indicated ratio. Surviving target cells were evaluated by flow cytometry at 48 hours. UTD: Untransduced T cells; Trast: Trastuzumab-based CAR. [Figure 5]214D8 p95HER2 CAR T design, expression, and cytotoxicity. (A) Schematic diagram of a chimeric receptor containing scFv that binds to full-length HER2 or p95HER2. (B) Surface expression of the indicated CAR on T cells in A 5 days post-transduction; the percentage of positive CAR T cells from total T cells is shown. (C) MCF10A p95HER2 cells were co-cultured with CAR T cells in the indicated ratio. After 48 hours, viable target cells were evaluated by flow cytometry. UTD: untransduced T cells; Trast: trastuzumab-based CAR. [Figure 6] Design, expression, and cytotoxicity of humanized 214D8 p95HER2 CAR T cells. (A) Schematic diagram of the chimeric receptor containing scFv that binds to p95HER2. (B) Surface expression of the indicated CAR on T cells in A 5 days post-transduction; the percentage of positive CAR T cells from total T cells is shown. (C) MCF10A p95HER2 cells or MCF10A wild-type cells were co-cultured with CAR T cells in the indicated ratio. After 48 hours, viable target cells were evaluated by flow cytometry. UTD: Untransduced T cells. [Figure 7] Design, expression, and cytotoxicity of 215C2 p95HER2 CAR T cells. (A) Schematic diagram of a chimeric receptor containing scFv that binds to full-length HER2 or p95HER2. (B) Surface expression of the indicated CAR on T cells 5 days post-transduction; the percentage of positive CAR T cells from total T cells is shown. (C) MCF10A p95HER2 cells were co-cultured with CAR T cells in the indicated ratio. After 48 hours, viable target cells were evaluated by flow cytometry. UTD: Untransduced T cells; Trast: Trastuzumab-based CAR. [Figure 8]Design, expression, and cytotoxicity of humanized 215C2 p95HER2 CAR T cells. (A) Schematic diagram of the chimeric receptor containing scFv that binds to p95HER2. (B) Surface expression of the indicated CAR on T cells in A 5 days post-transduction; the percentage of positive CAR T cells from total T cells is shown. (C) MCF10A p95HER2 cells or MCF10A wild-type cells were co-cultured with CAR T cells in the indicated ratio. After 48 hours, viable target cells were evaluated by flow cytometry. UTD: Untransduced T cells. [Figure 9] Efficacy of m215-derived p95HER2 CAR T cells on the proliferation of p95HER2-positive tumors in vivo. (A) MCF7 p95HER2 cells were orthotopically transplanted into mice. When the tumors reached approximately 300 mm3, they were treated with 3 × 10⁶ CAR+ T cells. (B) Percentage of circulating human CD3+ T cells per μl of blood relative to total leukocytes at day 144. [Figure 10-1] Specific antitumor effects of h1_214-derived p95HER2 CAR T cells against the growth of p95HER2-positive (MCF7p95HER2) and p95HER2-negative (MCF7) tumors in vivo. Mice were orthotopically transplanted with MCF7p95HER2 cells (A) or MCF7 cells (D). When the tumor reached approximately 300 mm3, the mice were treated with 3 × 10⁶ CAR+ T cells or UTD T cells by tail vein injection, and 10 days later they received a second dose with the same number of T cells. The number of circulating human CD3+ T cells per μl of blood (B, E) was determined 10 days after administration of the second dose. The number of tumor-infiltrating CD3 cells per milligram of tumor was evaluated at the indicated time points (C, F). [Figure 10-2]Specific antitumor effects of h1_214-derived p95HER2 CAR T cells against the growth of p95HER2-positive (MCF7p95HER2) and p95HER2-negative (MCF7) tumors in vivo. Mice were orthotopically transplanted with MCF7p95HER2 cells (A) or MCF7 cells (D). When the tumor reached approximately 300 mm3, the mice were treated with 3 × 10⁶ CAR+ T cells or UTD T cells by tail vein injection, and 10 days later they received a second dose with the same number of T cells. The number of circulating human CD3+ T cells per μl of blood (B, E) was determined 10 days after administration of the second dose. The number of tumor-infiltrating CD3 cells per milligram of tumor was evaluated at the indicated time points (C, F). [Figure 11-1] The complete amino acid sequence of p95HER2-CAR disclosed in this document. [Figure 11-2] The complete amino acid sequence of p95HER2-CAR disclosed in this document. [Figure 12] Summary of the scFv amino acid sequence. [Modes for carrying out the invention]

[0022] This invention relates to providing novel compounds for the treatment of cancer.

[0023] Unless otherwise defined, all technical terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention belongs.

[0024] All embodiments and definitions disclosed in connection with one aspect of the present invention are also applicable to other aspects of the present invention.

[0025] Chimeric antigen receptor (CAR) In the first aspect, the present invention is (i) A p95HER2-specific antigen-binding domain, (ii) Transmembrane domain and (iii) relating to a chimeric antigen receptor (CAR) comprising at least one intracellular signaling domain and / or a costimulatory domain, The antigen-binding domain is, (i) -The framework areas of the VL and VH domains have been humanized, - The VH region contains the sequences of sequence numbers 1, 2, and 3 or their functional equivalent variants, or the sequences of sequence numbers 1, 174, and 3 or their functional equivalent variants, -ScFv, characterized in that CDR1, CDR2, and CDR3 in the VL region each contain the sequences of SEQ ID NOs. 4, 5, and 6 or their functional equivalent variants, or the sequences of SEQ ID NOs. 175, 5, and 6 or their functional equivalent variants, (ii) - Having at least one VH region and at least one VL region, -At least CDR1, CDR2, and CDR3 in the VH region contain the sequences of sequence numbers 7, 8, and 9, or their functionally equivalent variants, -An antigen-binding domain (antigen-binding domain 1) characterized in that at least the VL region CDR1, CDR2, and CDR3 each contain the sequence of SEQ ID NOs. 10, 11, and 12 or a functionally equivalent variant thereof, and (iii) - Having at least one VH region and at least one VL region, - At least one VH region CDR1, CDR2, and CDR3 each contain the sequence of sequence numbers 13, 14, and 15 or their functionally equivalent variants, - Selected from the group consisting of antibodies or antibody fragments, characterized in that at least one VL region CDR1, CDR2, and CDR3 each contain the sequence of SEQ ID NOs. 16, 17, and 18 or a functionally equivalent variant thereof, or the sequence of SEQ ID NOs. 179, 17, and 18 or a functionally equivalent variant thereof.

[0026] As used herein, “chimeric antigen receptor (CAR),” also known as a chimeric T cell receptor, T-isomer, artificial T cell receptor, and chimeric immune receptor (CIR) are engineered receptors that transfer arbitrary specificity onto immune effector cells. In classical CARs, the specificity of a monoclonal antibody is transferred onto T cells. Thus, a CAR is a fusion protein comprising at least an extracellular domain or antigen-binding domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide on which the extracellular domain is induced, and at least one intracellular costimulatory domain.

[0027] According to the present invention, the terms “extracellular domain,” “antigen-binding domain,” “antigen-binding fragment,” or “antibody fragment” are used interchangeably and refer to any oligopeptide or polypeptide capable of binding to a particular antigen. It may include an antibody fragment, which refers to at least one portion of an intact antibody, or a recombinant variant thereof, for example, an antigen-variable region of an antibody that is intact enough to enable recognition and specific binding of the antibody fragment to a target. The antigen-binding domain of the present invention includes at least VH and VL regions. Examples of antibody fragments, but not limited to these, include Fab, Fab'-, F(ab')2, and Fv fragments, ScFv antibody fragments, and linear antibodies. In the context of the present invention, an antigen-binding domain or antibody fragment includes at least one VH region and one VL region, but may include two VL regions and two VH regions. Therefore, for example, in one embodiment, the antigen-binding domain is ScFv and therefore includes only one VL region and one VH region. In another embodiment, the antigen-binding domain is a Fab fragment, which in this case comprises one VL and VH (Fab or Fab') or two VH and two VL regions (Fab2 or F(ab')2).

[0028] In certain embodiments, the antigen-binding domain is humanized.

[0029] As used herein, the “humanized” form of a non-human (e.g., mouse) antibody or antigen-binding domain is a chimeric antibody or antigen-binding domain that contains or does not contain the smallest sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody or antigen-binding domain is a human immunoglobulin (recipient antibody), where residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capability. In some examples, Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody or antigen-binding domain may contain residues not found in the recipient antibody or donor antibody. These modifications are generally made to further improve the performance of the antibody or antigen-binding domain. Generally, a humanized antibody or antigen-binding domain contains substantially all of at least one and typically two variable domains, in which case all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FR residues are from a human immunoglobulin sequence. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0030] The antigen-binding domain of the CAR of the present invention specifically recognizes the carboxyl-terminal fragments of HER2 and p95HER2.

[0031] The terms “HER2” and “HER2 receptor” are used interchangeably herein and refer to the ErbB2 protein (also referred to as HER2 / neu in the literature). As used herein, the terms are intended to include variants (e.g., splice variants), isoforms, and homologs (both orthologs and paralogs) of HER2. In some embodiments, binding of the anti-HER2 binding molecules disclosed herein to HER2 inhibits the proliferation of HER2-expressing cells (i.e., typically tumor cells, and in particular cancer cells expressing low levels of HER2) by inhibiting the formation of heteromer complexes between HER2 and other ErbB family members, for example, by inhibiting heterodimerization with EGFR or HER3.

[0032] HER2 is a receptor tyrosine kinase and consists of an extracellular domain (ECD) comprising (i) two leucine-rich domains (domain I / L1 and domain III / L2) involved in ligand binding, and (ii) two cysteine-rich domains (domain II / CR1 and domain IV / CR2) involved in receptor dimerization; a transmembrane domain; and an intracellular tyrosine kinase domain. Alternative splice variants of HER2 exist and may be part of the present invention.

[0033] As used herein, the term “p95HER2” refers to the carboxy-terminal fragment (CTF) of the HER2 receptor protein, also known as “611-CTF” or “100–115kDa p95HER2”. The p95HER2 fragment is generated in cells through the initiation of translation of HER2 mRNA at codon position 611 of the full-length HER2 molecule (Anido et al, EMBO J 25;3234-44 (2006)). It has a molecular weight of 100–115kDa and is expressed at the cell membrane, where it can form homodimers maintained by intermolecular disulfide bonds.

[0034] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs) or complementarity-determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0035] As used herein, the terms “hypervariable region,” “HVR,” “complementarity-determining region,” or “CDR” refer to each region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop (hypervariable loop). Generally, natural quadruple-chain antibodies contain six CDRs: three in the VH chain (H1, H2, H3) and three in the VL chain (L1, L2, L3). Thus, the CDRs determine the affinity (primarily binding strength) and specificity of a protein to a particular antigen. The CDRs of the two chains in each pair are aligned by a framework region and acquire the function of binding to a specific epitope. As a result, both the heavy and light chains are characterized by three CDRs, VH-CDR1, VH-CDR2, VH-CDR3 and VL-CDR1, VL-CDR2, VL-CDR3, respectively.

[0036] The CDR sequence can be determined according to conventional criteria, for example, by means of the criteria in IgBLAST: http: / / www.ncbi.nlm.nih.gov / igblast / (Ye et al., 2013, Nucleic Acids Res 41 (Web Server issue: W34-40), according to the numbering provided by Kabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), or according to the numbering provided by Chothia et al. (1989, Nature 342: 877-83). This particular region can be determined by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), and Chothia et al. As described by al., J.Mol.Biol.196:901-917(1987), the definition includes duplicates or subsets of amino acid residues when compared to one another. The exact number of residues that constitute a particular CDR varies depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues constitute a particular CDR given the variable region amino acid sequence of an antibody. The CDR sequences given herein generally follow the Kabat definition.

[0037] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0038] In certain embodiments, the antigen-binding domain of the CAR of the present invention is ScFv.

[0039] As used herein, “single-stranded variable fragment (ScFv)” means a single-stranded polypeptide derived from an antibody that retains the ability to bind to an antigen. Examples of ScFv include antibody polypeptides formed by recombinant DNA technology, in which the variable (Fv) regions of immunoglobulin heavy chain (VH chain) and light chain (VL chain) fragments are linked via spacer sequences. Various methods for preparing ScFv are known, including those described in U.S. Patent No. 4,694,778, Nature, vol. 334, p. 54454 (1989), and Science, vol. 242, pp. 1038-1041 (1988).

[0040] The second element of the CAR according to the present invention is a transmembrane domain that is bound to the extracellular domain of the CAR.

[0041] As used herein, “transmembrane domain” (TMD) refers to a region of a CAR that transverses the cell membrane. The transmembrane domain of the CAR of the present invention is a transmembrane domain of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids related to the extracellular region of the protein in which transmembrane is induced (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 of the extracellular region) and / or one or more additional amino acids related to the intracellular region of the protein in which the transmembrane protein is induced (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to up to 15 of the intracellular region). In one embodiment, the transmembrane domain is associated with one of the other domains of the CAR used. In some cases, transmembrane domains can be selectively or modified by amino acid substitutions to avoid binding of the same or different surface membrane proteins to such domains, for example, to minimize interaction with other members of the receptor complex. In one embodiment, the transmembrane domain can homodimerize with another CAR on the surface of the CART cell. In a different embodiment, the amino acid sequence of the transmembrane domain may be modified or substituted to minimize interaction with the binding domain of a native binding partner present in the same CART.

[0042] The transmembrane domain may originate from either a natural or recombinant source. If the source is natural, the domain may originate from any membrane-binding protein or transmembrane protein. In one embodiment, the transmembrane domain can signal to the intracellular domain whenever the CAR binds to a target. Examples of specific uses or transmembrane domains in the present invention include, for example, alpha, beta, or zeta chains of T cell receptors, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD3 zeta, KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, CTLA4, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFl), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4 CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDGA, CDGA, CD103, ITGAL, CDLa, LFA-1, ITGAM, CDllb, ITGAX, CDlc, ITGB1, CD29, ITGB2, CD18, LFA-1, LGA ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT It may include transmembrane regions of transmembrane domains selected from the transmembrane domains of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A), Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, Kp30, NKp46 (including NKG2D), and / or NKG2C.

[0043] In a particular embodiment, the transmembrane domain is selected from the group consisting of the CD4 transmembrane domain, the CD8 transmembrane domain, the CD28 transmembrane domain, the 4-1BB transmembrane domain, the CTLA4 transmembrane domain, the CD27 transmembrane domain, and the CD3 zeta transmembrane domain.

[0044] In certain embodiments, the transmembrane domain is the CD28 transmembrane domain. In certain embodiments, the CD28 transmembrane domain contains the sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 113).

[0045] The CAR according to the present invention comprises at least one intracellular signaling domain and / or a co-stimulatory domain.

[0046] Where used herein, “intracellular signaling domain” refers to the intracellular portion of a molecule, and more specifically, to any oligopeptide or polypeptide known to function as a domain that transmits signals causing activation or inhibition of intracellular biological processes. An intracellular signaling domain generates signals that stimulate the immune effector function of CAR-containing cells, e.g., CAR-T cells. The effector function of a T cell may be, for example, cytolytic function including cytokine secretion or helper activity. Therefore, an intracellular signaling domain may be part of a protein that transduces effector function signals and directs a cell (e.g., a T cell) to perform a specialized function.

[0047] In general, entire intracellular signaling domains may be used; however, it is understood that it is not necessary to use the entire domain, provided that any part of the signaling domain used can still transduce the effector functional signal. It will also be understood that variants of such intracellular signaling domains having substantially the same or greater functional capacity may be used. This implies that the variant should have substantially the same or greater transduction of the effector functional signal. Typically, substantially the same or greater signaling includes at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120%, or more, of the signaling of the unmodified intracellular signaling domain, with the signaling of the unmodified intracellular signaling domain accounting for 100%. Methods for evaluating the transduction of effector functional signals are well known to those skilled in the art and include, for example, evaluating the quantity and / or activity of the transduced signaling molecule (e.g., a protein such as a cytokine). Therefore, if the signal is the cytolytic function of T cells, the method may involve measuring one or more cytokines secreted by T cells, which are known to have cytolytic activity (e.g., I₂FN-gamma). Another means of evaluating cytolytic function is, as is well known in the art, by CFSE staining and counting of positive cells by flow cytometry or chromium release assay.

[0048] Examples of intracellular signaling domains for use in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and coreceptors that act jointly after engagement with antigen receptors to initiate signal transduction, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities.

[0049] Signals generated solely via the TCR are generally insufficient for complete T cell activation, and secondary and / or co-stimulatory signals may also be required. Therefore, T cell activation can be said to be mediated by two distinct classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling domain), and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary intracellular signaling domains such as the co-stimulatory domain). The co-stimulatory domain may promote the activation of effector function and also promote the persistence of effector function and / or cell survival.

[0050] The primary intracellular signaling domain modulates the primary activation of the TCR complex either stimulatingly or inhibitorily. A primary intracellular signaling domain acting stimulatingly may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or ITAMs (e.g., two, three, four, five, or more ITAMs). Therefore, an intracellular signaling domain may contain one or more ITAMs. Examples of ITAMs containing primary intracellular signaling domains particularly used in this invention include CD3 zeta, Fc receptor gamma, Fc receptor beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0051] In one embodiment, the intracellular signaling domain of the CAR of the present invention is CD3 zeta, and more specifically, the CAR of the present invention comprises the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 114).

[0052] The terms “zeta” or alternatively “zeta chain,” “CD3 zeta,” or “TCR zeta” are defined as the protein represented by GenBank entry number BAG36664.1, or equivalent residues from non-human species such as mice, rodents, monkeys, or primates, and the “zeta-stimulating domain” or alternatively “CD3 zeta-stimulating domain” or “TCR zeta-stimulating domain” are defined as amino acid residues of the cytoplasmic domain of the zeta chain that are sufficient for the functional transmission of the primary signal required to activate T cells, etc. In one embodiment, the zeta cytoplasmic domain includes residues 52-164 of the GenBank entry protein BAG36664.1, or equivalent residues from non-human species, such as mice, rodents, monkeys, or primates, which are their functional orthologists.

[0053] It will be understood that one or more ITAMs in the intracellular signaling domain can be modified, for example, by mutation. Modifications can be used to increase or decrease the signaling function of the ITAM compared to the native ITAM domain.

[0054] As described above, an intracellular signaling domain may contain a primary intracellular signaling domain by itself, or it may contain a primary intracellular signaling domain in combination with one or more secondary intracellular signaling domains, such as one or more co-stimulatory signaling domains. Therefore, the intracellular signaling domain of CAR may contain a CD3 zeta signaling domain by itself, or in combination with one or more other intracellular signaling domains, such as one or more co-stimulatory signaling domains.

[0055] The co-stimulus signaling domain refers to the CAR portion of a co-stimulus molecule that includes the intracellular domain.

[0056] The term "costimulatory molecule" refers to a recognizable T cell-binding partner that mediates a costimulatory response exerted by T cells, such as proliferation, by specifically binding to a costimulatory ligand, although these molecules are not limited to these. Costimulatory molecules are cell surface molecules other than antigen-specific receptors or their ligands that are necessary for an effective immune response. Costimulatory molecules may be cell surface molecules other than antigen receptors or their ligands that are required for an efficient response by immune cells (e.g., lymphocytes) to an antigen. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules (SLAM proteins), and NK cell activation receptors. Examples of such molecules, but are not limited to these, are listed in 4-1. Examples include ligands that specifically bind to BB(CD137), OX40, ICOS, DAP10, CD27, CD28, CDS, CD30, CD137(4-1BB), CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, GITR, NKG2C, SLAMF7, NKp80, BAFFR, HVEM, BTLA, ICAM-1, LFA-1(CD11a / CD18), B7-H3, and CD83. For example, CD27 costimulation has been shown to enhance the proliferation, effector function, and survival of human CART cells in vitro, and to increase the persistence and antitumor activity of human T cells in vivo (Song et al. Blood. 2012;1 19(3):696-706).

[0057] In certain embodiments, the CAR of the present invention comprises the intracellular domain of the co-stimulatory molecule CD28, and more specifically, the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 115).

[0058] In a preferred embodiment, at least one intracellular signaling domain includes a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0059] In another embodiment, at least one intracellular signaling domain includes an intracellular domain of a co-stimulatory molecule selected from OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

[0060] In certain embodiments, at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0061] In another embodiment, at least one intracellular signaling domain is located N-terminally relative to the CD3 zeta intracellular domain. In yet another embodiment, at least one intracellular signaling domain is the intracellular domain of the co-stimulatory molecule CD28, located N-terminally relative to the CD3 zeta intracellular domain.

[0062] The intracellular signaling sequences within the intracellular portion of the CAR of the present invention can be linked to each other randomly or in a specific order. Optionally, short oligolinkers or polypeptide linkers of, for example, 2 to 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) can form links between the intracellular signaling sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In another embodiment, a single amino acid such as alanine or glycine can be used as a suitable linker.

[0063] In one embodiment, the intracellular signaling domain is designed to include two or more, for example, three, four, five, or more, co-stimulatory signaling domains. In one embodiment, two or more, for example, two, three, four, five, or more, co-stimulatory signaling domains are separated by a linker molecule, such as those described herein. In one embodiment, the intracellular signaling domain includes two co-stimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.

[0064] In a preferred embodiment, the intracellular portion of the CAR is -CD3 zeta signaling domain and CD28 signaling domain, -The signaling domain of CD3 zeta and the signaling domain of 4-1 BB, -CD3 zeta signaling domain and OX40 signaling domain, -CD3 zeta signaling domain and ICOS signaling domain, -CD3 zeta signaling domain and DAP10 signaling domain, -Includes the signaling domain of CD3 zeta, the signaling domain of 4-1 BB, and the signaling domain of OX40. -4-1 Signaling domain of BB zeta and signaling domain of CD28.

[0065] In another embodiment, the intracellular portion of the CAR includes a CD3 zeta signaling domain, a 4-1 BB signaling domain, and a CD28 signaling domain.

[0066] An intracellular signaling domain may include the entire intracellular portion, the entire intrinsic intracellular signaling domain, the molecule from which it originates, or a functional fragment thereof.

[0067] The antigen-binding domain of the CAR of the present invention is selected from ScFv1 and two antigen-binding domains, antigen-binding domain 1, and antigen-binding domain 2.

[0068] ScFv1 is, -V L and V H The domain has been humanized, -V H The regions CDR1, CDR2, and CDR3 each contain the sequences of sequence numbers 1, 2, and 3 or their functional equivalent variants, or the sequences of sequence numbers 1, 174, and 3 or their functional equivalent variants, -VL The CDR1, CDR2, and CDR3 of the region each contain the sequence of SEQ ID NO: 4, 5, and 6 or a functionally equivalent variant thereof, or the sequence of SEQ ID NO: 175, 5, and 6 or a functionally equivalent variant thereof.

[0069] In certain embodiments, the V L region of ScFv1 is located at the N-terminus or C-terminus relative to the V H region. In a more preferred embodiment, the V L region of ScFv1 is located at the N-terminus relative to the V H region.

[0070] The antigen-binding domain 1 having at least one VH region and at least one VL region - The CDR1, CDR2, and CDR3 of at least one V H region each contain the sequence of SEQ ID NO: 7, 8, and 9 or a functionally equivalent variant thereof. - The CDR1, CDR2, and CDR3 of at least the V L region each contain the sequence of SEQ ID NO: 10, 11, and 12 or a functionally equivalent variant thereof.

[0071] The antigen-binding domain 2 having at least one VH region and at least one VL region - The CDR1, CDR2, and CDR3 of the V H region each contain the sequence of SEQ ID NO: 13, 14, and 15 or a functionally equivalent variant thereof. - The CDR1, CDR2, and CDR3 of the V L region each contain the sequence of SEQ ID NO: 16, 17, and 18 or a functionally equivalent variant thereof, or the sequence of SEQ ID NO: 179, 17, and 18 or a functionally equivalent variant thereof.

[0072] As used herein, the term “functional equivalent variant of a CDR sequence” refers to a sequence variant of a particular CDR sequence that has substantially similar sequence identity to that particular CDR sequence and substantially maintains its ability to bind to its homologous antigen when it is part of an antibody, antibody fragment, or antigen-binding domain as an ScFv as described herein. For example, a functional equivalent variant of a CDR sequence may be a polypeptide sequence derivative of that sequence, comprising the addition, deletion, or substitution of one or more amino acids. In one embodiment, the substitution of one amino acid by the other in the functional equivalent variant is a conservative substitution.

[0073] As used herein, the term “conservative substitution” refers to the substitution of an amino acid with another amino acid having similar chemical properties. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions of each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0074] A functionally equivalent variant of a CDR sequence according to the present invention includes a CDR sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding amino acid sequence shown in one of the above reference sequences. The functionally equivalent variant of a CDR sequence is also intended to include an addition of at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, or more amino acids at the N-terminus, or at the C-terminus, or at both the N-terminus and C-terminus of the corresponding amino acid sequence shown in one of the above reference sequences. Similarly, the variant is also intended to include a deletion at the N-terminus, or at the C-terminus, or at both the N-terminus and C-terminus of the corresponding amino acid sequence shown in one of the sequences described above, consisting of at least one amino acid, or at least two amino acids, or at least three amino acids, or at least four amino acids, or at least five amino acids, or at least six amino acids, or at least seven amino acids, or at least eight amino acids, or at least nine amino acids, or at least ten amino acids, or more amino acids.

[0075] A functionally equivalent variant of a CDR sequence according to the present invention, preferably being part of an antibody fragment such as the ScFv of the CAR of the present invention or part of an antigen-binding domain, maintains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200%, or more of the ability of the corresponding amino acid sequence shown in one of SEQ ID NOs. 1-18 and 174-179 to bind to its congeneral antigen. This ability to bind to its congeneral antigen can be determined as a value of affinity, affinity, specificity, and / or selectivity of the antibody or antibody fragment against that congeneral antigen.

[0076] In certain embodiments, FR1, FR2, FR3, and FR4 of the VH region of ScFv1 each contain the sequences of sequence numbers 19, 20, 21, and 22 or their functionally equivalent variants, and FR1, FR2, FR3, and FR4 of the VL region of ScFv1 each contain the sequences of sequence numbers 23, 24, 25, and 26 or their functionally equivalent variants.

[0077] In another specific embodiment, FR1, FR2, FR3, and FR4 of at least one VH region of antigen-binding domain 1 each comprise the sequences of SEQ ID NOs. 31, 32, 33, and 34 or functionally equivalent variants thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of antigen-binding domain 1 each comprise the sequences of SEQ ID NOs. 35, 36, 37, and 38 or functionally equivalent variants thereof.

[0078] In another embodiment, FR1, FR2, FR3, and FR4 of at least one VH region of antigen-binding domain 1 each include the sequence of SEQ ID NOs. 31, 32, 33, and 34, SEQ ID NOs. 65, 66, 67, and 68, or SEQ ID NOs. 73, 74, 75, and 76, or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of antigen-binding domain 1 each include SEQ ID NOs. 35, 36, 37, and 38, SEQ ID NOs. 69, 70, 71, and 72, or SEQ ID NOs. 77, 78, 79, and 80, or a functionally equivalent variant thereof.

[0079] In another specific embodiment, FR1, FR2, FR3, and FR4 of at least one VH region of antigen-binding domain 2 each comprise the sequences of SEQ ID NOs. 42, 43, 44, and 45 or functionally equivalent variants thereof, and FR1, FR2, FR3, and FR4 of at least VL region of antigen-binding domain 2 each comprise the sequences of SEQ ID NOs. 46, 47, 48, and 49 or functionally equivalent variants thereof.

[0080] In another embodiment, FR1, FR2, FR3, and FR4 of at least one VH region of antigen-binding domain 2 each include the sequence of SEQ ID NOs. 42, 43, 44, and 45, SEQ ID NOs. 89, 90, 91, and 92, or SEQ ID NOs. 97, 98, 99, and 100, or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of the antigen each include the sequence of SEQ ID NOs. 46, 47, 48, and 49, or SEQ ID NOs. 93, 94, 95, and 96, or a functionally equivalent variant thereof.

[0081] As used herein, the term “functional equivalent variant of an FR sequence” refers to a sequence variant of a particular FR sequence that has substantially similar sequence identity to that particular FR sequence and substantially maintains its ability to bind to a cognate antigen when it is part of an antibody or antibody-binding domain described herein. For example, a functional equivalent variant of an FR sequence may be a polypeptide sequence derivative of said sequence, comprising the addition, deletion, or substitution of one or more amino acids.

[0082] A functionally equivalent variant of an FR sequence according to the present invention includes an FR sequence having at least approximately 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the corresponding amino acid sequence shown in one of the above reference sequences. The functionally equivalent variant of an FR sequence is also intended to include an addition of at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, at least ten amino acids, or more amino acids at the N-terminus, or at the C-terminus, or at both the N-terminus and C-terminus of the corresponding amino acid sequence shown in one of the above reference sequences. Similarly, the variant is also intended to include a deletion at the N-terminus, or at the C-terminus, or at both the N-terminus and C-terminus of the corresponding amino acid sequence shown in one of the sequences described above, consisting of at least one amino acid, or at least two amino acids, or at least three amino acids, or at least four amino acids, or at least five amino acids, or at least six amino acids, or at least seven amino acids, or at least eight amino acids, or at least nine amino acids, or at least ten amino acids, or more amino acids.

[0083] A functionally equivalent variant of an FR sequence according to the present invention, preferably when it is part of the antigen-binding domain of the present invention, maintains at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 200%, or more of the ability of the corresponding amino acid sequence shown in one of SEQ ID NOs. 19-26, 31-38, and 42-49 to bind to its congener antigen. This ability to bind to its congener antigen can be determined as the affinity, affinity, specificity, and / or selectivity of the antibody or antibody fragment against that congener antigen.

[0084] In one embodiment, VL of ScFv1 includes the sequence of sequence number 27 or 180 or its functionally equivalent variant, and VH of ScFc1 includes the sequence of sequence number 28 or 181 of its functionally equivalent variant.

[0085] In one embodiment, at least one VL of antigen-binding domain 1 includes the sequence of SEQ ID NO: 39 or a functionally equivalent variant thereof, and VH of antigen-binding domain 1 includes the sequence of SEQ ID NO: 40 or a functionally equivalent variant thereof.

[0086] In another embodiment, at least one VL region of antigen-binding domain 1 comprises the sequence of SEQ ID NO: 39, 54, or 56 or a functionally equivalent variant thereof, and at least one VH region of antigen-binding domain 1 comprises the sequence of SEQ ID NO: 40, 53, or 55 or a functionally equivalent variant thereof.

[0087] In one embodiment, at least one VL of the antigen-binding domain 2 includes the sequence of SEQ ID NO: 50 or 184 or a functionally equivalent variant thereof, and at least one VH of the antigen-binding domain 2 includes the sequence of SEQ ID NO: 51 or a functionally equivalent variant thereof.

[0088] In another embodiment, at least one VL region of antigen-binding domain 2 includes the sequence of SEQ ID NO: 50, 184, 60, or 62, and at least one VH region of antigen-binding domain 2 includes a sequence selected from SEQ ID NO: 51, 59, or 61 or a functionally equivalent variant thereof.

[0089] Preferred embodiments of the VL and VH regions of ScFv1 are defined below.

[0090] 1. In one embodiment, the VL of ScFv1 according to the present invention is characterized as follows: 1.1. The CDR1 region does not contain the sequence KASQNVGTAVA (sequence number 10 or 16), and optionally it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% with the sequence KASQSVGTAVA (sequence number 4) or the sequence RASQSVGTAVA (sequence number 175). 1.2. The CDR1 region does not contain an Asn residue at position 5, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% with the sequence KASQSVGTAVA (SEQ ID NO: 4) or RASQSVGTAVA (SEQ ID NO: 175). 1.3. The CDR1 region differs from the sequence of KASQSVGTAVA (SEQ ID NO: 4) or the sequence RASQSVGTAVA (SEQ ID NO: 175) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids, and optionally, it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with the sequence KASQSVGTAVA (SEQ ID NO: 4) or the sequence RASQSVGTAVA (SEQ ID NO: 175). 1.4. The CDR1 region does not contain the sequence KASQNVGTAVA (SEQ ID NO: 10 or 16), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids, or all of them, are conservative substitutions of amino acids found in the sequence KASQSVGTAVA (SEQ ID NO: 4) or at positions corresponding to the sequence RASQSVGTAVA (SEQ ID NO: 175). 1.5. The CDR1 region does not contain the sequence KASQNVGTAVA (SEQ ID NO: 10 or 16), and it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids in common with the sequence KASQSVGTAVA (SEQ ID NO: 4) or the sequence RASQSVGTAVA (SEQ ID NO: 175).

[0091] 2. In one embodiment, the VL of ScFv1 according to the present invention is characterized as follows: 2.1. The CDR2 region does not contain the sequence SASNRYT (sequence number 11 or 17), and optionally it exhibits sequence identity with the sequence SASNRFT (sequence number 5) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.2. The CDR2 region does not contain a Tyr residue at position 6, and optionally, it exhibits sequence identity with sequence SASNRFT (Sequence ID 5) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.3. The CDR2 region differs from the sequence of SASNRFT (SEQ ID NO: 5) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.4. The CDR2 region does not contain the sequence SASNRYT (sequence number 11 or 17), and at least one, at least two, at least three, at least four, at least five, or all of the amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence SASNRFT (sequence number 5). 2.5. The CDR2 region does not contain the sequence SASNRYT (sequence number 11 or 17), and it contains at least one, at least two, at least three, at least four, or at least five amino acids in common with the sequence SASNRFT (sequence number 5).

[0092] 3. In one embodiment, the VL of ScFv1 according to the present invention is characterized as follows: 3.1. The CDR3 region does not contain a sequence containing the sequence QQYSTYPLT (sequence number 12) or the sequence QQYSSYPLT (sequence number 18), and optionally it exhibits sequence identity with sequence QQYSTYPLA (sequence number 6) of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99%. 3.2. The CDR3 region does not contain a Thr residue at position 9 and / or a Ser residue at position 5, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% with sequence QQYSTYPLA (SEQ ID NO: 6). 3.3. The CDR3 region differs from the sequence of QQYSTYPLA (SEQ ID NO: 6) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 3.4. The CDR3 region does not contain the sequence QQYSTYPLT (SEQ ID NO: 12) or the sequence QQYSSYPLT (SEQ ID NO: 18), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence QQYSTYPLA (SEQ ID NO: 6). 3.5. The CDR3 region does not contain the sequence QQYSTYPLT (SEQ ID NO: 12) or the sequence QQYSSYPLT (SEQ ID NO: 18), and it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight amino acids in common with the sequence of QQYSTYPLA (SEQ ID NO: 6).

[0093] 4. In one embodiment, the VH of ScFv1 according to the present invention is characterized as follows: 4.1. The CDR1 region does not contain the sequence TYGMA (SEQ ID NO: 7) or the sequence DYGMS (SEQ ID NO: 13), and optionally, it exhibits sequence identity with the sequence DFGMS (SEQ ID NO: 1) of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99%. 4.2. The CDR1 region does not contain a Thr residue at position 1, a Tyr residue at position 2, and / or an Ala residue at position 5, and optionally it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with sequence DFGMS (Sequence ID 1). 4.3. The CDR1 region differs from the sequence of DFGMS(SEQ ID NO: 1) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 4.4. The CDR1 region does not contain the sequence TYGMA (SEQ ID NO: 7) or the sequence DYGMS (SEQ ID NO: 13), and at least one, at least two, at least three, at least four, or at least five amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence DFGMS (SEQ ID NO: 1). 4.5. The CDR1 region does not contain the sequence TYGMA (SEQ ID NO: 7) or the sequence DYGMS (SEQ ID NO: 13), and it contains at least one, at least two, at least three, or at least four amino acids in common with the sequence of DFGMS (SEQ ID NO: 1).

[0094] 5. In one embodiment, the VH of ScFv1 according to the present invention is characterized as follows: 5.1. The CDR2 region does not contain the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8) or the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14), and optionally, it exhibits at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% sequence identity with the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2) or the sequence TINTNGGTTHYPDSVKG (SEQ ID NO: 174). 5.2. The CDR2 region does not contain a Ser or Gly residue at position 4, a Val residue at position 7, a Lys residue at position 8, an Ile residue at position 9, a Tyr residue at position 10, a His residue at position 11, or a Val residue at position 12, and, optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2) or the sequence TINTNGGTTHYPDSVKG (SEQ ID NO: 174). 5.3. The CDR2 region differs from the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 8) or the sequence TINTNGGTTHYPDSVKG (SEQ ID NO: 14) by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 amino acids. And optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% with sequence TINTNGGTTHYPDNVKG (Sequence ID 2) or sequence TINTNGGTTHYPDSVKG (Sequence ID 174). 5.4. The CDR2 region does not contain the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8) or the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen amino acids, or all amino acids, are conservative substitutions of amino acids found at the corresponding positions in the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2) or the sequence TINTNGGTTHYPDSVKG (SEQ ID NO: 174). 5.5. The CDR2 region does not contain the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8) or the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14), and it contains, in common with the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2) or the sequence TINTNGGTTHYPDSVKG (SEQ ID NO: 174), at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 16 amino acids, and contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids.

[0095] 6. In one embodiment, the VH of ScFv1 according to the present invention is characterized as follows: 6.1. The CDR3 region does not contain the sequence EGFDY (sequence number 9 or 15), and optionally it exhibits sequence identity with sequence EGLDY (sequence number 3) of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99%. 6.2. The CDR3 region does not contain a Phe residue at position 3, and optionally, it exhibits sequence identity with sequence EGLDY (Sequence ID 3) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 6.3. The CDR3 region differs from the sequence of EGLDY (SEQ ID NO: 3) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 6.4. The CDR3 region does not contain the sequence EGFDY (sequence number 9 or 15), and at least one, at least two, at least three, at least four, or at least five amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence EGLDY (sequence number 3). 6.5. The CDR3 region does not contain the sequence EGFDY (sequence number 9 or 15), and it contains at least one, at least two, at least three, or at least four amino acids in common with the sequence EGLDY (sequence number 3).

[0096] Preferred embodiments of the VL and VH regions of antigen-binding domain 1 are defined below.

[0097] 1. In one embodiment, the VL of the antigen-binding domain 1 according to the present invention is characterized as follows: 1.1. The CDR1 region does not contain the sequence KASQSVGTAVA (sequence number 4), and optionally it exhibits sequence identity with sequence KASQNVGTAVA (sequence number 10) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 1.2. The CDR1 region does not contain a Ser residue at position 5, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% with the sequence KASQNVGTAVA (Sequence ID 10). 1.3. The CDR1 region differs from the sequence of KASQNVGTAVA (SEQ ID NO: 10) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 1.4. The CDR1 region does not contain the sequence KASQSVGTAVA (SEQ ID NO: 4), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids, or all of them, are conservative substitutions of amino acids found at the corresponding positions in the sequence KASQNVGTAVA (SEQ ID NO: 10). 1.5. The CDR1 region does not contain the sequence KASQSVGTAVA (SEQ ID NO: 4), and it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids in common with the sequence KASQNVGTAVA (SEQ ID NO: 10).

[0098] 2. In one embodiment, the VL of the antigen-binding domain 1 according to the present invention is characterized as follows: 2.1. The CDR2 region does not contain the sequence SASNRFT (sequence number 5), and optionally it exhibits sequence identity with the sequence SASNRYT (sequence number 11) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.2. The CDR2 region does not contain a Phe residue at position 6, and optionally, it exhibits sequence identity with sequence SASNRYT (Sequence ID 11) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.3. The CDR2 region differs from the sequence of SASNRYT (SEQ ID NO: 11) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.4. The CDR2 region does not contain the sequence SASNRFT (SEQ ID NO: 5), and at least one, at least two, at least three, at least four, at least five, or all of the amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence SASNRYT (SEQ ID NO: 11). 2.5. The CDR2 region does not contain the sequence SASNRFT (SEQ ID NO: 5), and it contains at least one, at least two, at least three, at least four, or at least five amino acids in common with the sequence SASNRYT (SEQ ID NO: 11).

[0099] 3. In one embodiment, the VL of the antigen-binding domain 1 according to the present invention is characterized as follows: 3.1. The CDR3 region does not contain the sequence QQYSTYPLA (sequence number 6) or the sequence QQYSSYPLT (sequence number 18), and optionally it exhibits sequence identity with sequence QQYSTYPLT (sequence number 12) of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99%. 3.2. The CDR3 region does not contain an Ala residue at position 9 and / or a serine residue at position 5, and optionally, it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with sequence QQYSTYPLT (SEQ ID NO: 12). 3.3. The CDR3 region differs from the sequence of QQYSTYPLT (Sequence ID 12) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 3.4. The CDR3 region does not contain the sequence QQYSTYPLA (SEQ ID NO: 6) or the sequence QQYSSYPLT (SEQ ID NO: 18), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence QQYSTYPLT (SEQ ID NO: 12). 3.5. The CDR3 region does not contain the sequence QQYSTYPLA (SEQ ID NO: 6) or the sequence QQYSSYPLT (SEQ ID NO: 18), and it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight amino acids in common with the sequence of QQYSTYPLT (SEQ ID NO: 12).

[0100] 4. In one embodiment, the VH of the antigen-binding domain 1 according to the present invention is characterized as follows: 4.1. The CDR1 region does not contain the sequence DFGMS (SEQ ID NO: 1) or the sequence DYGMS (SEQ ID NO: 13), and optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with the sequence TYGMA (SEQ ID NO: 7). 4.2. The CDR1 region does not contain an Asp residue at position 1, a Phe residue at position 2, and / or a Ser residue at position 5, and optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with sequence TYGMA (SEQ ID NO: 7). 4.3. The CDR1 region differs from the sequence of TYGMA (SEQ ID NO: 7) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 4.4. The CDR1 region does not contain the sequence DFGMS (SEQ ID NO: 1) or the sequence DYGMS (SEQ ID NO: 13), and at least one, at least two, at least three, at least four, or at least five amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence TYGMA (SEQ ID NO: 7). 4.5. The CDR1 region does not contain the sequence DFGMS or the sequence DYGMS, and it contains at least one, at least two, at least three, or at least four amino acids in common with the sequence of TYGMA (SEQ ID NO: 7).

[0101] 5. In one embodiment, the VH of the antigen-binding domain 1 according to the present invention is characterized as follows: 5.1. The CDR2 region does not contain the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2) or the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14), and optionally it exhibits sequence identity with the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8) of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99%. 5.2. The CDR2 region does not contain a Thr or Gly residue at position 4, a Val residue at position 7, a Thr residue at position 8, an Ile residue at position 9, a His residue at position 10, a Val residue at position 12, and / or an Asn residue at position 14, and optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with the sequence TINSNGGKTYHPDSVKG (Sequence ID 8). 5.3. The CDR2 region differs from TINSNNGKTYHPDSVKG (Sequence ID 8) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen amino acids, and optionally, it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with sequence TINSNNGKTYHPDSVKG (Sequence ID 8). 5.4. The CDR2 region does not contain the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2) or the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen amino acids, or all amino acids, are conservative substitutions of amino acids found at the corresponding positions in the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8). 5.5. The CDR2 region does not contain the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2) or the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14), and it contains, in common with the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8), at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 amino acids, and contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids.

[0102] 6. In one embodiment, the VH of the antigen-binding domain 1 according to the present invention is characterized as follows: 6.1. The CDR3 region does not contain the sequence EGLDY (sequence number 3), and optionally it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with the sequence EGFDY (sequence number 9) or the sequence DY. 6.2. The CDR3 region does not contain a Leu residue at position 3, and optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with sequence EGFDY (SEQ ID NO: 9) or sequence DY. 6.3. The CDR3 region differs from the sequence of EGFDY (SEQ ID NO: 9) or sequence DY by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with sequence EGFDY (SEQ ID NO: 9) or sequence DY. 6.4. The CDR3 region does not contain the sequence EGLDY (SEQ ID NO: 3), and at least one, at least two, at least three, at least four, or at least five amino acids are conservative substitutions of amino acids found in or at positions corresponding to the sequence DY in the sequence EGFDY (SEQ ID NO: 9). 6.5. The CDR3 region does not contain the sequence EGLDY (SEQ ID NO: 3), and it contains at least one, at least two, at least three, or at least four amino acids in common with the sequence EGFDY (SEQ ID NO: 9) or the sequence DY.

[0103] Preferred embodiments of the VL and VH regions of antigen-binding domain 2 are defined below.

[0104] 1. In one embodiment, the VL of the antigen-binding domain 2 according to the present invention is characterized as follows: 1.1. The CDR1 region does not contain the sequence KASQSVGTAVA (SEQ ID NO: 4), and optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with the sequence KASQNVGTAVA (SEQ ID NO: 16) or the sequence RASQNVGTAVA (SEQ ID NO: 179). 1.2. The CDR1 region does not contain a Ser residue at position 5, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% with the sequence KASQNVGTAVA (SEQ ID NO: 16) or RASQNVGTAVA (SEQ ID NO: 179). 1.3. The CDR1 region differs from the sequence of KASQNVGTAVA (SEQ ID NO: 16) or the sequence RASQNVGTAVA (SEQ ID NO: 179) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids, and optionally, it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with the sequence KASQNVGTAVA (SEQ ID NO: 16) or the sequence RASQNVGTAVA (SEQ ID NO: 179). 1.4. The CDR1 region does not contain the sequence KASQSVGTAVA (SEQ ID NO: 4), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids, or all of them, are conservative substitutions of amino acids found at the corresponding positions in the sequence KASQNVGTAVA (SEQ ID NO: 16) or the sequence RASQNVGTAVA (SEQ ID NO: 179). 1.5. The CDR1 region does not contain the sequence KASQSVGTAVA (SEQ ID NO: 4), and it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acids in common with the sequence KASQNVGTAVA (SEQ ID NO: 16) or the sequence RASQNVGTAVA (SEQ ID NO: 179).

[0105] 2. In one embodiment, the VL of the antigen-binding domain 2 according to the present invention is characterized as follows. 2.1. The CDR2 region does not contain the sequence SASNRFT (sequence number 5), and optionally it exhibits sequence identity with the sequence SASNRYT (sequence number 17) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.2. The CDR2 region does not contain a Phe residue at position 6, and optionally, it exhibits sequence identity with sequence SASNRYT (Sequence ID 17) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.3. The CDR2 region differs from the sequence of SASNRYT (SEQ ID NO: 17) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 2.4. The CDR2 region does not contain the sequence SASNRFT (SEQ ID NO: 5), and at least one, at least two, at least three, at least four, at least five, or all of the amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence SASNRYT (SEQ ID NO: 17). 2.5. The CDR2 region does not contain the sequence SASNRFT (SEQ ID NO: 5), and it contains at least one, at least two, at least three, at least four, or at least five amino acids in common with the sequence SASNRYT (SEQ ID NO: 17).

[0106] 3. In one embodiment, the VL of the antigen-binding domain 2 according to the present invention is characterized as follows: 3.1. The CDR3 region does not contain a sequence containing the sequence QQYSTYPLA (sequence number 6) or the sequence QQYSTYPLT (sequence number 12), and optionally it exhibits sequence identity with sequence QQYSSYPLT (sequence number 18) of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99%. 3.2. The CDR3 region does not contain an Ala residue at position 9 and / or a Thr residue at position 5, and optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with sequence QQYSSYPLT (Sequence ID 18). 3.3. The CDR3 region differs from the sequence of QQYSSYPLT (Sequence ID 18) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 3.4. The CDR3 region does not contain the sequence QQYSTYPLA (SEQ ID NO: 6) or the sequence QQYSTYPLT (SEQ ID NO: 12), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence QQYSSYPLT (SEQ ID NO: 18). 3.5. The CDR3 region does not contain the sequence QQYSTYPLA (SEQ ID NO: 6) or the sequence QQYSTYPLT (SEQ ID NO: 12), and it contains at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight amino acids in common with the sequence of QQYSSYPLT (SEQ ID NO: 18).

[0107] 4. In one embodiment, the VH of the antigen-binding domain 2 according to the present invention is characterized as follows: 4.1. The CDR1 region does not contain the sequence TYGMA (SEQ ID NO: 7) or the sequence DFGMS (SEQ ID NO: 1), and optionally, it exhibits sequence identity with the sequence DYGMS (SEQ ID NO: 13) of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99%. 4.2. The CDR1 region does not contain a Thr residue at position 1, a Phe residue at position 2, and / or an Ala residue at position 5, and optionally, it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with sequence DYGMS (SEQ ID NO: 13). 4.3. The CDR1 region differs from the sequence of DYGMS (SEQ ID NO: 13) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 4.4. The CDR1 region does not contain the sequence TYGMA (SEQ ID NO: 7) or the sequence DFGMS (SEQ ID NO: 1), and at least one, at least two, at least three, at least four, or at least five amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence DYGMS (SEQ ID NO: 13). 4.5. The CDR1 region does not contain the sequence TYGMA (SEQ ID NO: 7) or the sequence DFGMS (SEQ ID NO: 1), and it contains at least one, at least two, at least three, or at least four amino acids in common with the sequence of DYGMS (SEQ ID NO: 13).

[0108] 5. In one embodiment, the VH of the antigen-binding domain 2 according to the present invention is characterized as follows. 5.1. The CDR2 region does not contain the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8) or the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2), and optionally, it exhibits at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% sequence identity with the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14). 5.2. The CDR2 region does not contain a Ser or Thr residue at position 4, a Gly residue at position 7, a Thr residue at position 8, a Thr residue at position 9, a His residue at position 10, a His residue at position 11, a Pro residue at position 12, and / or an Asn residue at position 14, and optionally, it exhibits at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% sequence identity with the sequence TINGNGVKIYYVDSVKG (Sequence ID 14). 5.3. The CDR2 region differs from TINGNGVKIYYVDSVKG (Sequence ID 14) by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen amino acids, and optionally, it exhibits sequence identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% with sequence TINGNGVKIYYVDSVKG (Sequence ID 14). 5.4. The CDR2 region does not contain the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8) or the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2), and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen amino acids, or all amino acids, are conservative substitutions of amino acids found at the corresponding positions in the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14). 5.5. The CDR2 region does not contain the sequence TINSNGGKTYHPDSVKG (SEQ ID NO: 8) or the sequence TINTNGGTTHYPDNVKG (SEQ ID NO: 2), and it contains, in common with the sequence TINGNGVKIYYVDSVKG (SEQ ID NO: 14), at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 amino acids, and contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids.

[0109] 6. In one embodiment, the VH of the antigen-binding domain 2 according to the present invention is characterized as follows: 6.1. The CDR3 region does not contain the sequence EGLDY (sequence number 3), and optionally it exhibits sequence identity with sequence EGFDY (sequence number 15) of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 6.2. The CDR3 region does not contain a Leu residue at position 3, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99% with sequence EGFDY (Sequence ID 15). 6.3. The CDR3 region differs from the sequence of EGFDY (SEQ ID NO: 15) by at least one, at least two, at least three, at least four, or at least five amino acids, and optionally, it exhibits sequence identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or at least 99%. 6.4. The CDR3 region does not contain the sequence EGLDY (SEQ ID NO: 3), and at least one, at least two, at least three, at least four, or at least five amino acids are conservative substitutions of amino acids found at the corresponding positions in the sequence EGFDY (SEQ ID NO: 15). 6.5. The CDR3 region does not contain the sequence EGLDY (SEQ ID NO: 3), and it contains at least one, at least two, at least three, or at least four amino acids in common with the sequence EGFDY (SEQ ID NO: 15).

[0110] In another embodiment, ScFv1 includes the sequence of sequence number 30 or 182 or its functional equivalent.

[0111] In another embodiment, the antigen-binding domain 1 includes the sequence of SEQ ID NO: 41 or a functional equivalent thereof.

[0112] In another embodiment, the antigen-binding domain 1 includes the sequence of SEQ ID NOs: 41, 187, 188, or 189, or a functionally equivalent variant thereof.

[0113] In another embodiment, the antigen-binding domain 2 includes the sequence of SEQ ID NO: 52 or 186 or a functional equivalent thereof.

[0114] In another embodiment, the antigen-binding domain 2 includes the sequence of SEQ ID NOs. 52, 186, 190, or 191, or a functionally equivalent variant thereof.

[0115] In a particular embodiment, the VL of ScFv1 includes a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with sequence number 27 or 180. In a preferred embodiment, the VL of ScFv1 includes the sequence of sequence number 27 or 180, or a functionally equivalent variant having at least 85% sequence identity with sequence number 27 or 180.

[0116] In a particular embodiment, the VH of ScFv1 includes a sequence having at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity. In a preferred embodiment, the VH of ScFv1 includes the sequence of sequence number 28 or 181, or a functionally equivalent variant having at least 67% sequence identity with sequence number 28 or 181.

[0117] In certain embodiments, ScFv1 includes the sequence of sequence number 28, or a functionally equivalent variant thereof. In certain embodiments, ScFv1 includes a sequence having at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with sequence number 30 or 182. In preferred embodiments, ScFv1 includes the sequence of sequence number 30 or 182, or a functionally equivalent variant having at least 76% sequence identity with sequence number 30 or 182.

[0118] In another specific embodiment, at least one VL of antigen-binding domain 1 comprises the sequence of SEQ ID NO: 39, and at least one VH of antigen-binding domain 1 comprises the sequence of SEQ ID NO: 40 or a functionally equivalent variant thereof.

[0119] In certain embodiments, at least one VL of antigen-binding domain 1 contains a sequence having at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 39. In preferred embodiments, at least one VL of antigen-binding domain 1 contains the sequence of SEQ ID NO: 39, or a functionally equivalent variant having at least 39% sequence identity with SEQ ID NO: 39.

[0120] In certain embodiments, at least one VH of antigen-binding domain 1 contains a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 40. In preferred embodiments, at least one VH of antigen-binding domain 1 contains the sequence of SEQ ID NO: 40, or a functionally equivalent variant having at least 74% sequence identity with SEQ ID NO: 40.

[0121] In certain embodiments, antigen-binding domain 1 includes the sequence of SEQ ID NO: 41 or a functionally equivalent variant thereof. In other embodiments, antigen-binding domain 1 includes a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 41. In preferred embodiments, antigen-binding domain 1 includes the sequence of SEQ ID NO: 41 or a functionally equivalent variant having at least 85% sequence identity with SEQ ID NO: 41.

[0122] In another specific embodiment, at least one VL of antigen-binding domain 2 comprises the sequence of SEQ ID NO: 50 or 184 or a functional equivalent thereof, and at least one VH of antigen-binding domain 2 comprises the sequence of SEQ ID NO: 51 or a functional equivalent variant thereof.

[0123] In certain embodiments, at least one VL of the antigen-binding domain 2 contains a sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 50 or 184. In preferred embodiments, at least one VL of the antigen-binding domain contains the sequence of SEQ ID NO: 50 or 184, or a functionally equivalent variant having at least 89% sequence identity with SEQ ID NO: 50 or 184.

[0124] In certain embodiments, at least one VH of antigen-binding domain 2 contains a sequence having at least 74%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with SEQ ID NO: 51. In preferred embodiments, at least one VH of antigen-binding domain 2 contains the sequence of SEQ ID NO: 51, or a functionally equivalent variant having at least 67% sequence identity with SEQ ID NO: 51.

[0125] In certain embodiments, antigen binding 2 includes the sequence of SEQ ID NO: 52 or 186 or a functionally equivalent variant thereof. In other embodiments, antigen binding domain 2 includes a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 52 or 186. In preferred embodiments, antigen binding domain includes the sequence of SEQ ID NO: 52 or 186, or a functionally equivalent variant having at least 78% sequence identity with SEQ ID NO: 52 or 186.

[0126] In certain embodiments, the VH and / or VL regions of antigen-binding domains 1 and 2 of the CAR of the present invention are humanized.

[0127] Therefore, in certain embodiments, at least one VH region of antigen-binding domain 1 includes a humanized sequence selected from SEQ ID NOs. 53 and 55, or a functionally equivalent variant thereof, and at least one VL region of antigen-binding domain 1 includes a humanized sequence selected from SEQ ID NOs. 54 and 56, or a functionally equivalent variant thereof.

[0128] In certain embodiments, the antigen-binding domain 1 includes a humanized sequence selected from sequence numbers 187, 188, and 189.

[0129] In another embodiment, at least one VH and VL region of antigen-binding domain 1 comprises humanized FR1, FR2, FR3, and FR4 regions, wherein FR1, FR2, FR3, and FR4 of at least one VH region comprises the sequences of SEQ ID NOs. 65, 66, 67, and 68, respectively, or functionally equivalent variants thereof, and at least FR1, FR2, FR3, and FR4 of the VL region comprises the sequences of SEQ ID NOs. 69, 70, 71, and 72, respectively, or functionally equivalent variants thereof.

[0130] In another embodiment, at least one VH and VL region of antigen-binding domain 1 comprises humanized FR1, FR2, FR3, and FR4 regions, wherein FR1, FR2, FR3, and FR4 of at least one VH region comprises the sequences of SEQ ID NOs. 73, 74, 75, and 76, respectively, or functionally equivalent variants thereof, and FR1, FR2, FR3, and FR4 of at least one VL region comprises the sequences of SEQ ID NOs. 77, 78, 79, and 80, respectively, or functionally equivalent variants thereof.

[0131] In another embodiment, at least one VH region of the antigen-binding domain 1 of the CAR of the present invention comprises at least one humanized FR region, at least two humanized FR regions, at least three humanized FR regions, or at least four humanized FR regions, wherein the FR region is FR1: Sequence IDs 65 and 73; FR2: Sequence IDs 66 and 74; FR3: Sequence IDs 67 and 75; and FR4: Sequence IDs 68 and 76; Alternatively, it is selected from its functionally equivalent variants.

[0132] In another embodiment, at least one VL region of the antigen-binding domain 1 of the CAR of the present invention comprises at least one humanized FR region, at least two humanized FR regions, at least three humanized FR regions, or at least four humanized FR regions, wherein the FR region is FR1: Sequence IDs 69 and 77; FR2: Sequence IDs 70 and 78; FR3: Sequence IDs 71 and 79i; and FR4: Sequence IDs 72 and 80; Alternatively, it is selected from its functionally equivalent variants.

[0133] Similarly, in certain embodiments, at least one VH region of antigen-binding domain 2 comprises a humanized sequence selected from SEQ ID NOs. 59 and 61, or a functionally equivalent variant thereof, and at least one VH region of antigen-binding domain 1 comprises a humanized sequence selected from SEQ ID NOs. 60 and 62, or a functionally equivalent variant thereof.

[0134] In another embodiment, the antigen-binding domain 2 includes a humanized sequence selected from sequence numbers 190 and 191.

[0135] In another embodiment, at least one VH and VL region of antigen-binding domain 2 comprises humanized FR1, FR2, FR3, and FR4 regions, wherein FR1, FR2, FR3, and FR4 of at least one VH region comprises the sequences of SEQ ID NOs. 89, 90, 91, and 92, or functionally equivalent variants thereof, and FR1, FR2, FR3, and FR4 of at least one VL region comprises the sequences of SEQ ID NOs. 93, 94, 95, and 96, or functionally equivalent variants thereof.

[0136] In another embodiment, at least one VH and VL region of the antigen-binding domain 2 comprises humanized FR1, FR2, FR3, and FR4 regions, wherein FR1, FR2, FR3, and FR4 of at least one VH region comprises the sequences of SEQ ID NOs. 97, 98, 99, and 100, respectively, or functionally equivalent variants thereof, and FR1, FR2, FR3, and FR4 of at least one VL region comprises the sequences of SEQ ID NOs. 101, 102, 103, and 104, respectively, or functionally equivalent variants thereof.

[0137] In another embodiment, at least one VH region of the antigen-binding domain 2 of the CAR of the present invention comprises at least one humanized FR region, at least two humanized FR regions, at least three humanized FR regions, or at least four humanized FR regions. In some embodiments, the humanized FR region is selected from the group consisting of SEQ ID NOs: 89 and 97 for FR1, SEQ ID NOs: 90 and 98 for FR2, SEQ ID NOs: 91 and 99 for FR3, and SEQ ID NOs: 92 and 100 for FR4, or functionally equivalent variants thereof.

[0138] In another embodiment, at least one VL region of the antigen-binding domain 2 of the CAR of the present invention comprises at least one humanized FR region, at least two humanized FR regions, at least three humanized FR regions, or at least four humanized FR regions. In some embodiments, the humanized FR region is selected from the group consisting of SEQ ID NOs: 93 and 101 for FR1, SEQ ID NOs: 94 and 102 for FR2, SEQ ID NOs: 95 and 103 for FR3, and SEQ ID NOs: 96 and 104 for FR4, or their functionally equivalent variants.

[0139] In some examples, a CAR contains a linker between any two adjacent domains. For instance, a linker may be located between the transmembrane domain of an antigen-binding domain and a costimulatory domain. In another example, a linker may be located between an antigen-binding domain and an intracellular signaling domain.

[0140] In certain embodiments, when the antigen-binding domain is ScFv, the VH and VL regions of the antigen-binding domain are connected by a linker region containing Sequence ID No. 29.

[0141] In one embodiment, antigen-binding domain 1 is ScFv, and the VH and VL regions of ScFv are connected by a linker region containing sequence number 29. In another embodiment, antigen-binding domain 2 is ScFv, and the VH and VL regions of ScFv are connected by a linker region containing sequence number 29.

[0142] In certain embodiments, the linker is located between the VH and VL regions of ScFv. In even more specific embodiments, the linker between VH and VL includes the sequence of Sequence ID No. 29. In one embodiment, if the ScFv of the CAR of the present invention is ScFv1, then ScFv includes the structure VL-linker-VH or VH-linker-VL. In certain embodiments, if the ScFv of the CAR of the present invention is ScFv1, then ScFv includes the structure VL-linker-VH. In another embodiment, if antigen-binding domain 1 or 2 of the CAR of the present invention is ScFv, then ScFv may have the structure VH-linker-VL or VL-linker-VH. In certain embodiments, the linker is located C-terminally relative to the VL region and N-terminally relative to the VH region, i.e., VL-linker-VH.

[0143] The terms "flexible polypeptide linker" or "linker" refer to peptide linkers, consisting of amino acids such as glycine and / or serine residues, used alone or in combination to link a variable heavy chain region and a variable light chain region together, or to link any region or region of the CAR of the present invention.

[0144] Linker peptides can have any of a variety of amino acid sequences. Proteins can be linked by spacer peptides and generally have a flexible nature, but other chemical linkages are not excluded. Linkers can be peptides about 6 to 40 amino acids long, or about 6 to 25 amino acids long. These linkers can be made by linking proteins using synthetic linker-coding oligonucleotides. Peptide linkers with some degree of flexibility can be used. Linked peptides may have substantially any amino acid sequence, given that suitable linkers generally have sequences that result in flexible peptides. The use of small amino acids such as glycine and alanine is useful for making flexible peptides. The creation of such sequences is routine for those skilled in the art.

[0145] A suitable linker can be easily selected and may be any of several suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0146] Exemplary flexible linkers include a linker having the sequence TGSTSGSGKPGSGEGS (SEQ ID NO: 29). Preferred linkers include glycine polymers (G)n, glycine-serine polymers (e.g., including (GS)n, GSGGS n (SEQ ID NO: 117) and GGGS n (SEQ ID NO: 118), where n is at least one integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. In certain embodiments, the linker comprises a glycine polymer of formula (G4S)3. Glycine and glycine-serine polymers are interesting because both of these amino acids are relatively unstructured and can therefore act as neutral tethers between components. Glycine polymers are particularly interesting because glycine accesses significantly more phi-psi spaces than alanine and is far less restrictive than residues with longer side chains. Examples of flexible linkers include, but are not limited to, GGSG (SEQ ID NO: 119), GGSGG (SEQ ID NO: 120), GSGSG (SEQ ID NO: 121), GSGGG (SEQ ID NO: 122), GGGSG (SEQ ID NO: 123), and GSSSG (SEQ ID NO: 124). Those skilled in the art will recognize that the design of a peptide conjugated to any of the elements described above may include a linker that is all or partly flexible, thereby allowing the linker to include one or more parts that confer a flexible linker as well as a less flexible structure.

[0147] In another embodiment, the CAR of the present invention further comprises a hinge domain between the antigen-binding domain and the transmembrane domain.

[0148] As used herein, “hinge domain,” “hinge region,” or “spacer” refers to an amino acid region that enables the binding site and the separation and flexibility of the T cell membrane. The length of the flexible hinge also allows for better binding to relatively inaccessible epitopes; for example, a longer hinge domain allows for optimal binding. Those skilled in the art will be able to determine the appropriate hinge for a given CAR target.

[0149] In some examples, the first polypeptide of the CAR according to the present invention includes a hinge domain, which is sandwiched between an antigen-binding domain and a transmembrane domain. In some examples, the hinge domain is an immunoglobulin heavy chain hinge domain. In some examples, the hinge domain is a domain region polypeptide derived from a receptor (e.g., a CD8-derived hinge domain).

[0150] The hinge domain may have a length of approximately 10 to 200 amino acids, preferably 50 to 150 amino acids, and more preferably 75 to 125 amino acids.

[0151] Exemplary spacers include glycine polymers (G)n, glycine-serine polymers (e.g., including (GS)n, (GSGGS)n (SEQ ID NO: 125) and (GGGS)n (SEQ ID NO: 126), where n is at least one integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used, as both Gly and Ser are relatively unstructured and can therefore act as neutral tethers between components. Glycine polymers can be used, as glycine accesses significantly more phi-psi space than alanine and is far less restrictive than residues with longer side chains. Exemplary spacers may include, but are not limited to, amino acid sequences, including GGSG (SEQ ID NO: 127), GGSGG (SEQ ID NO: 128), GSGSG (SEQ ID NO: 129), GSGGG (SEQ ID NO: 130), GGGSG (SEQ ID NO: 131), GSSSG (SEQ ID NO: 132), and others.

[0152] In certain embodiments, the spacer includes the amino acid sequence GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116).

[0153] In some examples, the hinge domain in the first polypeptide of the CAR according to the present invention contains at least one cysteine. For example, in some examples, the hinge domain may contain the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 133). If present, the cysteine ​​in the hinge domain of the first CAR may be available to form a disulfide bond with the hinge domain of the second CAR.

[0154] The amino acid sequences of immunoglobulin hinge domains are known in the art; see, for example, Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162 and Huck et al. (1986) Nucl. Acids Res. 14:1779. As a non-limiting example, an immunoglobulin hinge domain may contain one of the following amino acid sequences: DKTHT (SEQ ID NO: 134); CPPC (SEQ ID NO: 133); CPEPKSCDTPPPCPR (SEQ ID NO: 136) (see, for example, Glaser et al. (2005) J. Biol. Chem. 280:41494); ELKTPLGDTTHT (SEQ ID NO: 137); KSCDKTHTCP (SEQ ID NO: 138); KCCVDCP (SEQ ID NO: 139); KYGPPCP (SEQ ID NO: 140); EPKSCDKTHTCPPCP (SEQ ID NO: 141) (Human IgG1 hinge); ERKCCVECPPCP (SEQ ID NO: 142) (Human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 143) (Human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 144) (Human IgG4 hinge), etc.

[0155] The hinge domain may contain the amino acid sequence of human IgG1, IgG2, IgG3, or IgG4. Compared to the wild-type (naturally occurring) hinge domain, the hinge domain may contain one or more amino acid substitutions and / or insertions and / or deletions. For example, His229 in the human IgG1 hinge can be substituted with Tyr, thereby the hinge domain containing the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 145). See, for example, Yan et al. (2012) J. Biol. Chem. 287:5891.

[0156] The hinge domain may contain an amino acid sequence derived from human CD8, for example, the hinge domain may contain the following amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 146), or a variant thereof.

[0157] In certain embodiments, the hinge domain is a CD8 hinge domain.

[0158] In another embodiment, the CAR of the present invention comprises, from N-terminus to C-terminus, an anti-p95HER2 light chain variable domain, a linker domain, an anti-p95HER2 heavy chain variable domain, a CD8 hinge domain, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, followed by a CD3 zeta intracellular signaling domain.

[0159] In certain embodiments, the hinge domain is a CD8 hinge domain, the transmembrane domain is a CD28 transmembrane domain, and the intracellular signaling domain is a CD28 co-stimulatory domain.

[0160] In certain embodiments, the CAR of the present invention comprises a CD8 hinge domain, a CD28 transmembrane domain, a CD3 zeta intracellular signaling domain, and a CD28 co-stimulatory domain.

[0161] Nucleic acids and host cells related to car of the present invention In a second aspect, the present invention relates to a nucleic acid encoding the CAR of the present invention.

[0162] This disclosure provides nucleic acids comprising nucleotide sequences encoding any of the CARs of the present invention.

[0163] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in either single-stranded or double-stranded forms. Unless specifically limited, the terms encompass nucleic acids including known analogues of native nucleotides that have similar binding capacity to that of a reference nucleic acid and are metabolized in the same way as naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implies conservatively modified variants (e.g., substitutions at degenerate codons), alleles, orthologs, SNPs, and complementary sequences, as well as sequences shown in their direct form. In particular, substitutions at degenerate codons can be obtained by constructing sequences in which the third position of one or more selected (or all) codons is replaced by a residue having a mixed base and / or a deoxyinosine residue.

[0164] In some cases, the nucleic acid of the subject provides the production of CARs of this disclosure, for example, in mammalian cells. In other cases, the nucleic acid of the subject provides amplification of CAR-coding nucleic acids.

[0165] A nucleotide sequence encoding any of the CARs of the present invention can be operably ligated to transcriptional regulatory elements, such as promoters and enhancers.

[0166] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambdaP, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements, cytomegalovirus immediate early promoter, herpes simplex virus thymidine kinase promoter, early and late SV40 promoters, promoters present in retroviral long-terminal repeats, mouse metallothionein-I promoter, and various tissue-specific promoters known in the art.

[0167] Suitable reversible promoters, including reversible inducible promoters, are known in the art. Such reversible promoters can be isolated and induced from many organisms, such as eukaryotes and prokaryotes. Modifications of reversible promoters derived from a first organism for use in a second organism, such as a first prokaryote and a second eukaryote, or a first eukaryote and a second prokaryote, are well known in the art. Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulating promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, alcohol transactivator protein (AlcR) responsive promoters, etc.), tetracycline-regulating promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulating promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal-regulating promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulatory promoters (e.g., salicylic acid-regulating promoters, ethylene-regulating promoters, benzothiadiazole-regulating promoters, etc.), temperature-regulating promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoters, etc.), light-regulating promoters, synthesis-inducible promoters, etc.).

[0168] In some cases, a locus or construct or transgene containing a suitable promoter is irreversibly switched by induction of an inducible system. Suitable systems for inducing irreversible switches are well known in the art, and for example, induction of an irreversible switch may utilize Cre-lox-mediated recombination. Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc., known in the art, can be used to generate irreversibly switchable promoters. Methods, mechanisms, and requirements for performing site-directed recombination, as described elsewhere herein, have found use in generating irreversibly switched promoters and are well known in the art.

[0169] In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used. For example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the Ncr1(p46) promoter; see, for example, Eckelhart et al. (2011) Blood 117:1565.

[0170] In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, and PYK1 promoter; or controllable promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHO5 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (for example, for use in Pichia). The selection of suitable vectors and promoters is well within the scope of those skilled in the art.

[0171] Promoter suitable for use in prokaryotic host cells includes, but is not limited to, the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, e.g., the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, etc.; the araBAD promoter; in vivo regulatory promoters, e.g., the ssaG promoter or related promoters, the pagC promoter, the nirB promoter, etc.; sigma 70 promoters, e.g., the consensus sigma 70 promoter; stationary phase promoters, e.g., the dps promoter, the spv promoter, etc.; promoters derived from pathogenic island SPI-2; the actA promoter; the rpsM promoter; the tet promoter; the SP6 promoter, etc. Potent promoters suitable for use in prokaryotes such as Escherichia coli include, but is not limited to, Trc, Tac, T5, T7, and P Lambda. Non-limiting examples of operators for use in bacterial host cells include lactose promoter operators (where the LacI repressor protein changes its conformation upon contact with lactose, thereby preventing the LacI repressor protein from binding to the operator), tryptophan promoter operators (where the TrpR repressor protein, when conjugated with tryptophan, has a conformation that binds to the operator; and where the TrpR repressor protein has a conformation that does not bind to the operator in the absence of tryptophan), and tac promoter operators.

[0172] In certain embodiments, the nucleic acid encoding the CAR of the present invention further comprises a sequence encoding a leader sequence that, after nucleic acid expression, provides a signal sequence located at the N-terminus of the CAR.

[0173] As used herein, the term "leader peptide" is used according to its ordinary meaning in the art and refers to a peptide having a length of about 5 to 30 amino acids. The leader peptide is present at the N-terminus of a newly synthesized protein that forms part of the secretory pathway. Proteins in the secretory pathway include, but are not limited to, proteins that are present within any of certain organelles (endoplasmic reticulum, Golgi apparatus, or endosome), secreted from the cell, or inserted into the cell membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of the protein.

[0174] In some embodiments, the isolated nucleic acid encodes a protein from the N-terminus to the C-terminus, and the leader peptide is present at the N-terminus of a newly synthesized protein that forms part of the secretory pathway. Proteins in the secretory pathway include, but are not limited to, proteins that are present within any of certain organelles (endoplasmic reticulum, Golgi apparatus, or endosome), secreted from the cell, or inserted into the cell membrane. In some embodiments, the leader peptide forms part of the transmembrane domain of the protein.

[0175] In some embodiments, the isolated nucleic acid encodes, from the N-terminus to the C-terminus, the protein: leader peptide, anti-p95HER2 light chain variable domain, linker domain, anti-p95HER2 heavy chain variable domain, CD8 hinge domain, CD28 transmembrane domain, CD28 intracellular co-stimulatory signaling domain, followed by the CD3 zeta intracellular signaling domain.

[0176] In another embodiment, the leader sequence is a CD8 leader sequence. In certain embodiments, the leader peptide comprises the sequence of SEQ ID NO: 147 (MALPVTALLLPLALLLHAARP).

[0177] In a third aspect, the invention relates to an expression vector comprising the nucleic acid of the invention.

[0178] As used herein, "vector", "cloning vector", and "expression vector" are vehicles by which a host is transformed, and expression (e.g., transcription and translation) of an introduced sequence means a vehicle that promotes introduction of a polynucleotide sequence (e.g., a foreign gene) into a host cell, and the vector includes plasmids, phages, viruses, etc.

[0179] A nucleotide sequence encoding any of the CARs of the present invention may be present in an expression vector and / or a cloning vector. The expression vector may include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, etc.

[0180] A number of suitable vectors and promoters are known to those skilled in the art, and many are commercially available for generating the recombinant constructs of the subject matter. The following vectors are provided as examples. Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0181] Expression vectors generally have a convenient restriction site located near the promoter sequence to provide insertion of a nucleic acid sequence encoding a heterologous protein. Selectable markers that can operate within the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and human immunodeficiency virus), and retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus, and retrovirus-derived vectors such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0182] As described above, in some embodiments, the nucleic acid containing any of the CARs of the present invention is, in some embodiments, RNA, for example, RNA synthesized in vitro. Methods for the in vitro synthesis of RNA are known in the art, and RNA containing nucleotide sequences encoding the first and / or second polypeptides of the heterodimer conditionally active CAR of the present disclosure can be synthesized using any known method. Methods for introducing RNA into host cells are known in the art. Introducing RNA containing nucleotide sequences encoding the first and / or second polypeptides of the heterodimer conditionally active CAR of the present disclosure into host cells can be carried out in vitro or ex vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo using RNA containing nucleotide sequences encoding the first and / or second polypeptides of the heterodimer conditionally active CAR of the present disclosure.

[0183] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also include either or both a selectable marker gene and / or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be supported on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as Neo. The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide that is either not present in the recipient organism or tissue, or is expressed by the recipient organism or tissue, and whose expression manifests as some readily detectable characteristic, such as enzyme activity. The expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, constructs having the smallest 5' facile region that exhibits the highest level of reporter gene expression are identified as promoters. Such promoter regions can be ligated to reporter genes and used to evaluate drugs for their ability to regulate promoter-driven transcription.

[0184] In a fourth aspect, the present invention relates to a host cell comprising the nucleic acid of the present invention or the expression vector of the present invention.

[0185] The terms “host cell” or “engineered cell” mean any cell of any organism that is modified, transformed, or engineered by the addition or modification of genes, DNA or RNA sequences, or proteins or polypeptides. It also refers to the offspring of such cells. The host cells or genetically engineered cells of the present invention include isolated immune cells such as T cells, NK cells, or NKT cells that contain DNA or RNA sequences encoding a chimeric antigen receptor or chimeric antigen receptor complex and express the chimeric receptor on their cell surface. Isolated host cells and engineered cells may be used, for example, to enhance NK or NKT cell activity or T lymphocyte activity, to treat cancer, and to treat infectious diseases.

[0186] In one embodiment, the cell containing any of the CAR polypeptides described herein, or the nucleic acid encoding any of the CAR polypeptides described herein, is a mammalian cell.

[0187] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, etc. Suitable mammalian cell lines, but not limited to these, include HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH This includes 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human fetal kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, and NK cell lines (e.g., NKL, NK92, and YTS).

[0188] In one embodiment, the mammalian cell comprises one of the CAR polypeptides described herein. The mammalian cell or tissue may be of human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cell may be used. In any preferred embodiment, the mammalian cell is human.

[0189] In some cases, the cells are not immortalized cell lines, but rather cells obtained from an individual (e.g., primary cells). For example, in some cases, the cells are immune cells obtained from an individual.

[0190] Manipulated cells may be obtained from peripheral blood, spinal blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue, or thymic tissue. Host cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. Cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and their transgenic species. Cells may be obtained from established cell lines.

[0191] The cells described above can be obtained by any known means. The cells may be self, syngeneic, homogeneous, or heterogeneous to the recipient of the manipulated cells. The term "self" refers to any material originating from the same individual that will later be reintroduced into the individual.

[0192] The term "homogeneous" refers to any material originating from different animals of the same species as the individual into which it is introduced. Two or more individuals are said to be homogeneous if one or more of their genes at one or more loci are not identical. In some aspects, homogeneous material originating from individuals of the same species may be genetically distinct enough to interact antigenically.

[0193] The term "heterogeneous" refers to grafts derived from animals of different species.

[0194] The term "homogeneous" refers to extremely close genetic similarity or identity, particularly with respect to an antigen or immune response. Homogeneous models include, for example, models in which organs and cells (e.g., cancer cells and their non-cancerous counterparts) originate from the same individual, and / or models in which organs and cells originate from different individual animals that belong to the same inbred lineage.

[0195] In one embodiment, the host cell is an immune cell.

[0196] As used herein, “immune cells” refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells; natural killer cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cells are T cells; NK cells; NKT cells; lymphocytes such as B cells and T cells; and myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0197] Immune cells can be obtained from subjects who have or have been diagnosed with cancer, plasma cell damage, or autoimmune disease or disorder. For example, immune cells can be obtained from subjects with cancer, such as multiple myeloma, smoldering myeloma, or Waldenström macroglobulinemia. In some embodiments, immune cells are obtained from subjects who are resistant to anti-BCMA therapy. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipient of the cells.

[0198] Immune cells (e.g., human immune cells) that can be used in the present invention include autologous cells obtained from the subject to whom the cells will be administered later after ex vivo modification and expansion. For example, immune cells can be obtained from an individual having or diagnosed with cancer, a plasma cell disorder, or an autoimmune disease or disorder. Immune cells can also be obtained from an allogeneic donor, which is a non-genetically identical individual of the same species as the intended recipient of the cells. Immune cells useful in the present invention include T cells and NK cells.

[0199] In another embodiment, the host cell is a T cell, a natural killer (NK) cell, or an NKT cell.

[0200] The terms "T cell" and "T lymphocyte" are interchangeable and are used interchangeably herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, or combinations thereof.

[0201] Natural killer cells or "NK cells" are well known in the art. In one embodiment, natural killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. NK cells can be detected by certain surface markers such as CD16, CD56, and CD8 in humans. NK cells do not express a T cell antigen receptor, the pan-T marker CD3, or a surface immunoglobulin B cell receptor.

[0202] NK cells mediate an anti-tumor effect without the risk of GvHD (graft-versus-host disease) and have a short lifespan compared to T cells. Thus, NK cells are depleted soon after destroying cancer cells, reducing the need for an inducible suicide gene on the CAR construct that excises the modified cells.

[0203] Natural killer T (NKT) cells are a heterogeneous group of T cells that share characteristics with both T cells and natural killer cells. Therefore, NKT cells are a subset of T cells that co-express the αβ T cell receptor, but also express various molecular markers typically associated with NK cells, such as NK1. Many of these cells recognize non-polymorphic CD1d molecules, which are antigen-presenting molecules that bind to self- and exogenous lipids and glycolipids. They constitute only about 0.1% of all peripheral blood T cells. Natural killer T cells should not be confused with natural killer cells.

[0204] In certain embodiments, T, NK, and NKT cells are derived from human peripheral blood mononuclear cells (PBMCs), leukocyte apheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord.

[0205] In one embodiment, the immune cells that may be used in the present invention (e.g., human immune cells) include autologous cells obtained from a subject to whom the cells will be subsequently administered, after ex vivo modification and expansion. For example, immune cells may be obtained from an individual that has or has been diagnosed with cancer. Immune cells may also be obtained from an allogeneic donor, which is a non-genetically identical individual of the same species as the intended recipient of the cells. Immune cells useful in the present invention include T, NK, and NKT cells.

[0206] Methods for obtaining T, NK, and NKT cells are known in the art and may be useful for the engineered immune cells described herein. T, NK, and NKT cells are typically obtained from peripheral blood collected from a subject, for example, by venipuncture or withdrawal via an implanted port or catheter. Optionally, blood can be obtained by a process including leukocyte apheresis, in which leukocytes are obtained from the subject's blood, while other blood components are returned to the subject. The blood or leukocyte apheresis product (fresh or cryopreserved) is processed to concentrate T, NK, or NKT cells using methods known in the art. For example, mononuclear cells (including T, NK, or NKT cells) can be concentrated by density gradient centrifugation (e.g., using Ficoll) and / or countercurrent centrifugation. For example, a T cell stimulation step can be performed on T cells to stimulate other cells, such as B cells, by using CD3 / CD28 antibodies coated on, for example, magnetic beads or artificial antigen-presenting cells (aAPCs) expressing, for example, cell surface-bound anti-CD3 and anti-CD28 antibody fragments (see below). The T cells in the enriched T cell preparation can then undergo genetic modification.

[0207] As an alternative to peripheral blood, tissues including bone marrow, lymph nodes, spleen, and tumors can be used as sources of T cells and NK cells. The T cells and NK cells may originate from humans, primates, hamsters, rabbits, rodents, cattle, pigs, sheep, horses, goats, dogs, or cats, but any other mammalian cells may be used. In certain embodiments of any configuration, the T or NK cells are human.

[0208] Immune cells such as T, NK, or NKT cells can be obtained from several sources including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spinal blood, thymic tissue, tissue from infection sites, ascites, pleural exudate, splenic tissue, and tumors. Any number of cell lines available in the art (e.g., immune cell lines such as T cell lines) may also be used.

[0209] In one embodiment, immune cells (e.g., T, NK, or NKT cells) are obtained from a unit of blood collected from a subject using any suitable technique known in the art, such as Ficoll® isolation. In another embodiment, cells derived from the circulating blood of a subject are obtained by apheresis. Apheresis products typically include lymphocytes, including T, NK, or NKT cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. It will be understood that cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps. For example, cells may be washed with phosphate-buffered saline (PBS). Alternatively, the washing solution may be calcium-deficient, magnesium-deficient, or lacking in many of them, not all divalent cations. An initial activation step in the absence of calcium may result in amplified activation. The washing step can be achieved by methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) in accordance with the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as Ca-free and Mg-free PBS, PlasmaLyte A, or other salines with or without buffer. Alternatively, the cells may be resuspended directly in the culture medium after removing any undesirable components from the apheresis sample.

[0210] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example by centrifugation with a PERCOLLTM gradient or by countercurrent centrifugation. Specific subpopulations of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, may be further isolated by positive or negative selection techniques known in the art. For example, T cells may be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3×28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells. Additionally or alternatively, T cell populations may be enriched by negative selection, for example by a combination of antibodies oriented to surface markers specific to negatively selected cells. Cell sorting and / or selection via negative magnetic immunoassay adherence or flow cytometry may be used.

[0211] It will be understood that cells derived from subjects modified to express the CAR of the present invention can be stored over a period prior to their use (see, for example, the therapeutic methods below). For example, cells may be optionally washed and then frozen, or incubated under conditions suitable for their survival until needed (e.g., on a rotator at 2–10°C or room temperature). In this way, cells can be stored until as needed. They may be stored in an unmodified state (i.e., without expressing the CAR of the present invention) or a modified state (i.e., modified to express the CAR of the present invention). Prior to use in the therapeutic applications further described below, cells may be activated and expanded using methods generally known in the art. For example, T cells may be expanded by contact with a surface bound to them, a drug that stimulates CD3 / TCR complex-associated signaling, and a ligand that stimulates costimulatory molecules on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, such as by contact with an anti-CD3 antibody, or its antigen-binding fragment, or an anti-CD2 antibody immobilized on its surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. Co-stimulation of accessory molecules on the surface of T cells is performed using ligands that bind to the accessory molecules. For example, T cell populations can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating T cell proliferation. Examples of anti-CD28 antibodies include 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), which can be used in other methods commonly known in the art.

[0212] T cells exposed to varying stimulation times may exhibit different characteristics. For example, typical blood or apherised peripheral blood mononuclear cell products contain a helper T cell population (TH, CD4+), which is larger than the cytotoxic or suppressive T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a T cell population in which TH cells become dominant before approximately 8-9 days, while after approximately 8-9 days, the T cell population includes an increasingly large TC cell population. Therefore, depending on the therapeutic objective, it may be advantageous to target and inject a T cell population predominantly containing TH cells. Similarly, if an antigen-specific subset of TC cells is isolated, it may be beneficial to expand this subset to a greater extent.

[0213] In certain embodiments, the T cells are CD8+ T cells.

[0214] In particular, the host cells of the present invention can be enlarged before transduction by the polynucleotide or vector of the present invention.

[0215] In a further aspect of the present invention, T cells are obtained from a patient immediately after treatment, leaving functional T cells in the subject. In this regard, it has been noted that after some cancer treatments, particularly treatment with drugs that damage the immune system, the quality of the T cells obtained shortly after treatment during the period when the patient should normally recover from treatment may be optimal or improved in relation to their ability to proliferate ex vivo. Also, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo proliferation. Therefore, in relation to the present invention, production of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic system, is provided during this stage of recovery. In addition, in some aspects, mobilization modes (e.g., mobilization using GM-CSF) and establishment of specific states may be used to create a state in a subject where regrowth, recirculation, regeneration, and / or reproduction of a particular cell type is advantageous, particularly within a certain time window after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0216] The manipulated cells described herein may also include suicide systems. Suicide systems provide a mechanism by which manipulated cells can be inactivated or destroyed, as described above. Such features allow for precise therapeutic control of any treatment in which manipulated cells are used. As used herein, suicide systems provide a mechanism by which cells having a suicide system can be inactivated or destroyed. Suicide systems are well known in the art.

[0217] In one embodiment, the suicide system includes a gene that can be pharmacologically activated to remove the containing cells if necessary. In certain embodiments, the suicide gene is not immunogenic to a host harboring polynucleotides or cells. In one example, the suicide system includes a gene that causes CD20 to be expressed on the cell surface of the manipulated cells. Therefore, the manipulated cells containing the gene may be destroyed using rituximab administration.

[0218] In some embodiments, the suicide system includes an epitope tag. Examples of epitope tags include the c-myc tag, the CD52 streptavidin-binding peptide (SBP), and the truncated EGFR gene (EGFRt). In this embodiment, the epitope tag is expressed in the manipulated cells. Therefore, the manipulated cells containing the gene may be destroyed by administering an antibody against the epitope tag.

[0219] In another embodiment, the suicide system includes a gene that causes the transection of epidermal growth factor receptors to be expressed on the surface of the engineered cells. Therefore, the engineered cells containing the gene may be destroyed using administration of cetuximab.

[0220] In another embodiment, the suicide system includes CD52 expressed on the surface of the manipulated cells. Therefore, the manipulated cells containing the gene may be destroyed using administration of an anti-52 ​​monoclonal antibody (CAMPATH, alemtuzumab).

[0221] In another embodiment, the suicide system includes CAMPATH (aremtuzumab). Therefore, the administration of an anti-52 ​​monoclonal antibody (CAMPATH) may be used to destroy the manipulated cells without expressing the tag or gene as CD52-highly expressing CAR T cells or T cells.

[0222] In another embodiment, the suicide gene may include a caspase 8 gene, a caspase 9 gene, a thymidine kinase, a cytosine deaminase (CD), or a cytochrome P450.

[0223] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred to host cells by physical, chemical, or biological means.

[0224] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0225] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0226] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). When non-viral delivery systems are utilized, the exemplary delivery vehicle is liposomes. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids may be associated with lipids. Lipid-associated nucleic acids may be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, bound to liposomes via linking molecules that associate with both liposomes and oligonucleotides, encapsulated within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as suspensions in lipids, containing micelles or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they may exist as micelles, in bilayer structures, or in "broken-down" structures. They may also simply be dispersed in solution and may form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds including long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0227] Suitable lipids can be obtained from commercially available sources. For example, dimyristiphosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, MO), dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristiphosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol.

[0228] "Liposome" is a general term encompassing various single and multilayer lipid vehicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an inner aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. The lipid components self-reorganize before the formation of a closed structure, encapsulating the solute dissolved between the water and the lipid bilayer. However, compositions having structures different from the usual vesicular structure in solution are also included. For example, lipids may assume a micelle structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0229] In some embodiments of this disclosure, any of the manipulated cells disclosed herein may be introduced by two vectors, each having a different CAR.

[0230] Regardless of the method used to introduce exogenous polynucleotides into host cells or otherwise expose cells to the polynucleotides of this disclosure, various assays may be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR, for example, by immunological means (ELSA and Western blotting) or by assays described herein, to identify drugs included within the scope of this disclosure; and “biochemical” assays, such as detecting the presence or absence of specific peptides.

[0231] The present invention includes ScFv, antigen-binding domain, and antibody. In a fifth aspect, the present invention is - The VH region contains the sequences of sequence numbers 1, 2, and 3 or their functional equivalent variants, or the sequences of sequence numbers 1, 174, and 3 or their functional equivalent variants, The present invention relates to an ScFv characterized in that CDR1, CDR2, and CDR3 of the VL region each contain the sequences of sequence numbers 4, 5, and 6 or their functional equivalent variants, or the sequences of sequence numbers 175, 5, and 6 or their functional equivalent variants.

[0232] In certain embodiments, FR1, FR2, FR3, and FR4 of the VH region of the ScFv of the present invention each comprise the sequences of SEQ ID NOs. 152, 153, 154, and 155 or their functionally equivalent variants, and FR1, FR2, FR3, and FR4 of the VL region of the ScFv or antigen-binding domain of the present invention each comprise the sequences of SEQ ID NOs. 156, 157, 158, and 159 or their functionally equivalent variants.

[0233] In another embodiment, FR1, FR2, FR3, and FR4 of the VH region of the ScFv of the present invention each include the sequence of sequence numbers 152, 153, 154, and 155, sequence numbers 19, 20, 21, and 22, or sequence numbers 163, 164, 165, and 166, or their functionally equivalent variants, and FR1, FR2, FR3, and FR4 of the VL region each include the sequence of sequence numbers 156, 157, 158, and 159, sequence numbers 23, 24, 25, and 26, or sequence numbers 167, 168, 169, or 170, or their functionally equivalent variants.

[0234] In another embodiment, VL of ScFv of the present invention includes the sequence of sequence number 160 or 193 or a functionally equivalent variant thereof, and VH of ScFc of the present invention includes the sequence of sequence number 161 or 194 of the functionally equivalent variant thereof.

[0235] In another embodiment, VL of the ScFv of the present invention includes the sequence of sequence numbers 160, 193, 27, 171, or 180 or a functionally equivalent variant thereof, and VH includes the sequence of sequence numbers 161, 194, 28, 172, or 181 or a functionally equivalent variant thereof.

[0236] In certain embodiments, the VH and VL regions of the ScFv of the present invention are connected by a linker region including sequence number 29.

[0237] In certain embodiments, the linker is located between the VH and VL regions of the antigen-binding domain. In one embodiment, ScFv may have the structure VH-linker-VL or VL-linker-VH. In certain embodiments, the linker is located C-terminally relative to the VL region and N-terminally relative to the VH region, i.e., VL-linker-VH.

[0238] In further embodiments, the ScFv of the present invention includes the sequence of sequence numbers 162 or 195 or functionally equivalent variants thereof.

[0239] In another embodiment, the ScFv of the present invention includes the sequence of sequence numbers 162, 195, 30, 173, or 182, or functionally equivalent variants thereof.

[0240] The definitions given within the context of the CAR of the present invention are equally applicable to the ScFv of the present invention. Similarly, possible functionally equivalent variants of the CDR that form part of the ScFv of the present invention provided herein have been previously defined and are equally applicable to the present example.

[0241] In a sixth aspect, the present invention is - Having at least one VH region and at least one VL region, - At least one VH region CDR1, CDR2, and CDR3 each contain the sequences of sequence numbers 7, 8, and 9 or their functionally equivalent variants, -The present invention relates to an antigen-binding domain characterized in that at least one VL region CDR1, CDR2, and CDR3 each contain the sequence of SEQ ID NOs. 10, 11, and 12 or a functionally equivalent variant thereof.

[0242] In certain embodiments, FR1, FR2, FR3, and FR4 of at least one VH region of the antigen-binding domain each include the sequences of SEQ ID NOs. 31, 32, 33, and 34 or functionally equivalent variants thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of the antigen-binding domain 1 each include the sequences of SEQ ID NOs. 35, 36, 37, and 38 or functionally equivalent variants thereof.

[0243] In another embodiment, FR1, FR2, FR3, and FR4 of at least one VH region of the antigen-binding domain of the present invention each comprise the sequence of SEQ ID NOs. 31, 32, 33, and 34, SEQ ID NOs. 65, 66, 67, and 68, or SEQ ID NOs. 73, 74, 75, and 76, or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of the antigen-binding domain of the present invention each comprise SEQ ID NOs. 35, 36, 37, and 38, SEQ ID NOs. 69, 70, 71, 72, or SEQ ID NOs. 77, 78, 79, and 80, or a functionally equivalent variant thereof.

[0244] In another embodiment, at least one VL of the antigen-binding domain of the present invention comprises the sequence of SEQ ID NO: 39 or a functionally equivalent variant thereof, and at least one VH of the ScFv of the present invention comprises the sequence of SEQ ID NO: 40 or a functionally equivalent variant thereof.

[0245] In another embodiment, at least one VL of the antigen-binding domain of the present invention comprises the sequences of SEQ ID NOs. 39, 54, and 56 or functionally equivalent variants thereof, and at least one VH region comprises the sequences of SEQ ID NOs. 40, 53, and 55 or functionally equivalent variants thereof.

[0246] In certain embodiments, when the antigen-binding domain is ScFv, the VH and VL regions are connected by a linker region containing SEQ ID NO: 29.

[0247] In certain embodiments, when the antigen-binding domain is ScFv, the linker is located between the VH region and the VL region. In one embodiment, when the antigen-binding domain 1 is ScFv, ScFv may have the structure VH-linker-VL or VL-linker-VH. In certain embodiments, when the antigen-binding domain is ScFv, the linker is located C-terminally relative to the VL region and N-terminally relative to the VH region, i.e., VL-linker-VH.

[0248] In further embodiments, the antigen-binding domain includes the sequence of SEQ ID NO: 41 or a functionally equivalent variant thereof.

[0249] In another embodiment, the antigen-binding domain of the present invention comprises the sequence of SEQ ID NOs: 41, 187, 188, or 189, or a functionally equivalent variant thereof.

[0250] The definitions given within the context of the CAR of the present invention apply equally to the antigen-binding domain of the present invention. Similarly, possible functionally equivalent variants of the CDR that form part of the antigen-binding domain of the present invention provided herein have been previously defined and are equally applicable to this example.

[0251] In the seventh aspect, the present invention is - Having at least one VH region and at least one VL region, - At least one VH region CDR1, CDR2, and CDR3 each contain the sequence of sequence numbers 13, 14, and 15 or their functionally equivalent variants, The present invention relates to an antibody or antibody fragment characterized in that at least one VL region CDR1, CDR2, and CDR3 each contain the sequence of SEQ ID NOs. 16, 17, and 18 or a functionally equivalent variant thereof, or the sequence of SEQ ID NOs. 179, 17, and 18 or a functionally equivalent variant thereof.

[0252] In certain embodiments, FR1, FR2, FR3, and FR4 of at least one VH region of the antibody or antibody fragment each comprise the sequence of SEQ ID NOs. 42, 43, 44, and 45 or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of the antibody or antibody fragment each comprise the sequence of SEQ ID NOs. 46, 47, 48, and 49 or a functionally equivalent variant thereof.

[0253] In another embodiment, FR1, FR2, FR3, and FR4 of at least one VH region of the antibody or antibody fragment of the present invention each include the sequence of SEQ ID NOs. 42, 43, 44, and 45, SEQ ID NOs. 89, 90, 91, and 92, or SEQ ID NOs. 97, 98, 99, and 100, or a functionally equivalent variant thereof, and FR1, FR2, FR3, and FR4 of at least one VL region of the antibody or antibody fragment of the present invention each include the sequence of SEQ ID NOs. 46, 47, 48, and 49, SEQ ID NOs. 93, 94, 95, and 96, or SEQ ID NOs. 101, 102, 103, and 104, or a functionally equivalent variant thereof.

[0254] In another embodiment, at least one VL of the antibody or antibody fragment comprises the sequence of SEQ ID NO: 50 or 184 or a functionally equivalent variant thereof, and at least one VH of the antibody or antibody fragment comprises the sequence of SEQ ID NO: 51 or a functionally equivalent variant thereof.

[0255] In another embodiment, at least one VL region of the antibody fragment comprises the sequence of SEQ ID NOs. 50 or 184, 60 or 62, or a functionally equivalent variant thereof, and at least one VH region of the antibody or the antibody fragment comprises the sequences of SEQ ID NOs. 51, 59, and 61, and / or functionally equivalent variants thereof.

[0256] In certain embodiments, if the antibody or antibody fragment is an ScFv, at least one VH and VL region of the antibody or antibody fragment is connected by a linker region containing SEQ ID NO: 29.

[0257] In certain embodiments, when the antibody or antibody fragment is an ScFv, the linker is located between the VH region and the VL region. In one embodiment, when the antibody or antibody fragment is an ScFv, the ScFv may have the structure VH-linker-VL or VL-linker-VH. In certain embodiments, when the antibody or antibody fragment is an ScFv, the linker is located C-terminus relative to the VL region and N-terminus relative to the VH region, i.e., VL-linker-VH.

[0258] In further embodiments, the antibody or antibody fragment comprises the sequence of SEQ ID NO: 52 or 186 or a functionally equivalent variant thereof.

[0259] In another embodiment, the antibody of the antibody fragment comprises the sequence of SEQ ID NOs. 52, 186, 190, or 191 or a functionally equivalent variant thereof.

[0260] As used herein, the term “antibody” refers to an immunoglobulin molecule, or, according to some embodiments of the present invention, a fragment of an immunoglobulin molecule having the ability to specifically bind to a molecular epitope (antigen). Naturally occurring antibodies typically comprise a tetramer consisting of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain, and is usually composed of three domains (CH1, CH2, and CH3). The heavy chain may be of any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes). Each light chain consists of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL). The light chain includes a kappa chain and a lambda chain. The heavy chain variable domains and light chain variable domains are typically involved in antigen recognition, while the heavy chain constant domains and light chain constant domains can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complementarity system (C1 q). The VH and VL domains can be further subdivided into hypervariable domains called "complementarity-determining regions," called "framework regions" (FRs), which contain interspersed domains of more conserved sequences. Each VH and VL consists of three CDR domains and four FR domains arranged from the amino terminus to the carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The heavy chain and light chain variable domains contain binding domains that interact with antigens. Of particular interest are antibodies and their epitope-binding fragments, which are "isolated" to exist in a physical environment distinct from those that may occur in nature, or which have been modified in their amino acid sequence to differ from naturally occurring antibodies.

[0261] The term "antibody" includes whole monoclonal or polyclonal antibodies, or fragments thereof, that possess one or more CDR regions, and includes human antibodies, humanized antibodies, chimeric antibodies, and antibodies of non-human origin.

[0262] A "monoclonal antibody" is a homogeneous, highly specific population of antibodies oriented to a single site or antigenic "determinant." A "polyclonal antibody" includes a heterogeneous population of antibodies oriented to different antigenic determinants.

[0263] In certain embodiments, the antibody of the present invention is a non-human antibody, preferably a mouse antibody. In preferred embodiments, the antibody of the present invention is a monoclonal antibody.

[0264] It is well known that the basic structural unit of an antibody is a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each consisting of a light chain (25 kDa) and a heavy chain (50-75 kDa). The amino-terminal region of each chain contains a variable region of approximately 100-110 or more amino acids involved in antigen recognition. The carboxy-terminal region of each chain contains a constant region that mediates effector function. The variable regions of each pair of light and heavy chains form the antibody binding site. Therefore, an intact antibody has two binding sites. The light chain is classified as K or λ. The heavy chain is classified as γ, μ, α, δ, and ε, which define the antibody isotype as IgG, IgM, IgA, IgD, or IgE, respectively.

[0265] The variable regions of each pair of light and heavy chains form the antibody binding sites. They are characterized by the same general structure, which consists of a relatively conserved region called a framework (FR) that is linked by three hypervariable regions called complementarity-determining regions (CDRs), as defined in the context of the extracellular domain or antigen-binding domain of the CAR of this invention.

[0266] Functional equivalent variants of the CDR and FR sequences that define the specificity of the antibody or antigen-binding domain of the present invention are intended herein. Accordingly, the definition of the functional equivalent variants of the sequences that define the CDR and FR of the antibody of the present invention, and the percentage of identity with respect to said sequences within the scope of the present invention, have already been defined in the context of the antigen-binding domain of the CAR of the present invention and apply equally to the antibody of the present invention.

[0267] Those skilled in the art will understand that the antibodies or antibody fragments of the present invention share all the features of the antigen-binding domain 2 of the CAR of the present invention, as this relates to their ability to bind to a specific antigen, namely the p95HER2 peptide. Therefore, all the details of the antigen-binding domain 2 of the CAR of the present invention relating to binding to the p95HER2 peptide apply to the antibodies or antibody fragments described herein (as they refer to their variable region).

[0268] As used herein, the antibodies of the present invention encompass not only full-length antibodies (e.g., IgG) but also their antigen-binding fragments, e.g., Fab, Fab', F(ab')2, Fv fragments, human antibodies, humanized antibodies, chimeric antibodies, antibodies of non-human origin, recombinant antibodies, and polypeptides derived from immunoglobulins produced by genetic engineering techniques, e.g., single-chain Fv(scFv), diabodies, heavy chains or fragments thereof, light chains or fragments thereof, VH or dimers thereof, VL or dimers thereof, Fv fragments stabilized by disulfide crosslinks (dsFv), molecules having single-chain variable region domains (Abs), minibodies, scFv-Fc, antibodies, fusion proteins with VL and VH domains, or any other modified configurations of immunoglobulin molecules containing antigen recognition sites of desired specificity. The antibodies of the present invention may also be bispecific antibodies. Antibody fragments may refer to antigen-binding fragments.

[0269] In certain embodiments, the antibody is selected from the group consisting of monoclonal antibodies, F(ab), F(ab'), Fv, ScFv, and minibodies.

[0270] As used herein, “recombinant antibody” is an antibody comprising amino acid sequences derived from two different species or two different sources, and includes synthetic molecules, such as antibodies comprising a non-human CDR and a human framework or constant region. In certain embodiments, the recombinant antibody of the present invention is produced from or synthesized from a recombinant DNA molecule.

[0271] Those skilled in the art will understand that the amino acid sequence of the antibody of the present invention may include one or more amino acid substitutions such that the primary sequence of the polypeptide is altered, but the ability of the antibody to bind to the p95HER antigen is maintained. Such substitutions may be conservative substitutions and are commonly used to indicate substitutions of one amino acid with another amino acid having similar properties (for example, the substitution of glutamic acid (a negatively charged amino acid) with aspartic acid is a conservative amino acid substitution).

[0272] The amino acid sequence modifications of antibodies described herein are intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies are prepared by introducing appropriate nucleotide changes into an antibody encoding a nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions is performed to achieve a final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter post-translational processes of the protein, such as changing the number or location of glycosylation sites.

[0273] Amino acid insertions include amino-terminus and / or carboxyl-terminus fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include peptides with an N-terminal methionyl residue, or antibody polypeptide chains fused to cytotoxic polypeptides. Other insertion variants of molecules include fusion to the N-terminus or C-terminus of enzymes or polypeptides that increase their serum half-life.

[0274] Another type of variant is amino acid substitution variant. These variants have at least one amino acid residue in the molecule that is replaced by a different residue. Hypervariability is the most targeted site for antibody substitution mutagenesis.

[0275] In certain embodiments, the ScFv, antigen-binding domain, and antibody or antibody fragment are humanized.

[0276] The term "humanization" has already been defined within the context of the CAR of the present invention and applies equally to this example. Similarly, preferred humanization sequences of the antigen-binding domain and antibody or antibody fragment (referring to its variable region, and therefore corresponding to antigen-binding domain 1 and antigen-binding domain 2, respectively) of the present invention have been previously defined within the context of the CAR of the present invention and apply equally to the antigen-binding domain or antibody or antibody fragment. Similarly, the ScFv of the present invention may be humanized. Therefore, in certain embodiments, the ScFv is humanized, and more specifically, the VH and / or VL regions of the ScFv are humanized.

[0277] In certain embodiments, the VL region of ScFv includes a humanized sequence selected from sequence numbers 27, 171, and 180, or a functionally equivalent variant thereof, and the VH region includes a humanized sequence selected from sequence numbers 28, 172, and 181, or a functionally equivalent variant thereof.

[0278] In another embodiment, ScFv includes a humanized sequence selected from sequence numbers 30, 173, and 182.

[0279] In another embodiment, the VH and VL regions of ScFv include humanized FR1, FR2, FR3, and FR4 regions, where FR1, FR2, FR3, and FR4 of the VH region each include the sequences of sequence numbers 19, 20, 21, and 22, or their functionally equivalent variants, and FR1, FR2, FR3, and FR4 of the VL region each include the sequences of sequence numbers 23, 24, 25, and 26, or their functionally equivalent variants.

[0280] In another embodiment, the VH and VL regions of ScFv include humanized FR1, FR2, FR3, and FR4 regions, where FR1, FR2, FR3, and FR4 of the VH region each include the sequences of sequence numbers 163, 164, 165, and 166, or their functionally equivalent variants, and FR1, FR2, FR3, and FR4 of the VL region each include the sequences of sequence numbers 167, 168, 169, and 170, or their functionally equivalent variants.

[0281] In another embodiment, the VH region of ScFv includes at least one humanized FR region, at least two humanized FR regions, at least three humanized FR regions, or at least four humanized FR regions. In yet another embodiment, the humanized FR1, FR2, FR3, and FR4 regions are selected from SEQ ID NOs. 19 or 163 for FR1, SEQ ID NOs. 20 or 164 for FR2, SEQ ID NOs. 21 or 165 for FR3, and SEQ ID NOs. 22 and 166 for FR4, or any of the functional equivalent variants described above.

[0282] In another embodiment, the VL region of ScFv includes at least one humanized FR region, at least two humanized FR regions, at least three humanized FR regions, or at least four humanized FR regions. In yet another embodiment, the humanized FR1, FR2, FR3, and FR4 regions are selected from SEQ ID NOs. 23 or 167 for FR1, SEQ ID NOs. 24 or 168 for FR2, SEQ ID NOs. 25 or 169 for FR3, and SEQ ID NOs. 26 and 170 for FR4, or any of the functional equivalent variants described above.

[0283] The present invention also provides derivatives of the ScFv, antigen-binding domain, or antibody disclosed herein. Derivatized ScFv, antigen-binding domain, or antibody may include any molecule or material that provides targeting properties, e.g., an increase in the half-life of the ScFv, antigen-binding domain, or antibody in a particular application. Derivatized ScFv, antigen-binding domain, or antibody may include molecules for special use (e.g., administration to a subject, e.g., human subject, or other in vivo or in vitro use), detectable (or labeled) residues (e.g., radioactive, colorimetric, antigenic, or enzyme molecules, detectable beads (e.g., magnetic or electron-density (e.g., gold) beads), or other molecules (e.g., biotin or streptavidin)), therapeutic or diagnostic residues (e.g., radioactive, cytotoxic, or pharmaceutically active residues), or molecules that increase the compatibility of the ScFv, antigen-binding domain, or antibody. Examples of molecules used to derivatize ScFv, antigen-binding domains, or antibodies include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-conjugated and pegylated derivatives of ScFv, antigen-binding domains, or antibodies can be prepared using techniques widely known in the art.

[0284] In some embodiments, the ScFv, antigen-binding domain, or antibody may contain one or more labels. “Label” means any detectable substance. Examples of suitable labels include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorescent substances), enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or certain polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags). In some embodiments, the label group is bound to the antibody via space arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art, and those skilled in the art will select appropriate labeling and methods for a particular purpose.

[0285] Generally, labels can be classified according to the detection method: a) radioactive or isotopic labels, b) magnetic labels (e.g., magnetic particles), c) redox active residues, d) optical dyes, enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinyl groups, and f) certain polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal-binding domains, epitope tags, etc.). In some embodiments, the labeling group is bound to the ScFv, antigen-binding domain, or antibody via spacer arms of varying lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art.

[0286] In one embodiment, the label includes, but is not limited to, an optical dye comprising a chromophore, a phosphor, and a fluorescent substance. The fluorescent substance may be a small molecule fluorescent substance or a protein fluorescent substance.

[0287] "Fluorescent labeling" refers to any molecule that can be detected by the fluorescent properties of a substance. Examples of fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, and BODIPY. Examples include, but are not limited to, FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon Green, alexa-fluor dyes (alexa-fluor 350, alexa-fluor 430, alexa-fluor 488, alexa-fluor 546, alexa-fluor 568, alexa-fluor 594, alexa-fluor 633, alexa-fluor 647, alexa-fluor 660, alexa-fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE), FITC), Cy5, Cy5.5, and Cy7.

[0288] Protein fluorescent labeling substances include, but are not limited to, green fluorescent proteins containing GFP species such as Renilla, Ptilosarcus, or Aequorea, EGFP (Clontech Labs., Inc., Genbank accession number U55762), blue fluorescent proteins, enhanced yellow fluorescent proteins, and β-galactosidase.

[0289] In the eighth aspect, the present invention relates to nucleic acids encoding ScFv, antigen-binding domains, or antibodies according to the fifth, sixth, and seventh aspects of the present invention.

[0290] In a ninth aspect, the present invention relates to an expression vector comprising a nucleic acid according to an eighth aspect of the present invention.

[0291] In a tenth aspect, the present invention relates to a host cell comprising a nucleic acid according to an eighth aspect of the present invention or an expression vector according to a ninth aspect of the present invention.

[0292] The definitions and details relating to the nucleic acids, expression vectors, and host cells associated with ScFv in this invention are the same as those defined within the context of CAR in this invention.

[0293] Diagnostic methods In the eleventh aspect, the present invention is (i) Contacting a patient sample containing tumor cells with the ScFv1, antigen-binding domain 1, or antibody of the present invention, (ii) A method for diagnosing cancer in a patient, comprising detecting the binding of ScFv, antigen-binding domains, or antibodies to cells in a sample, The presence of a bond indicates that the patient has cancer.

[0294] As used herein, the terms “cancer,” “tumor,” or “neoplastic disease” refer to a broad group of diseases involving unregulated cell proliferation, also known as malignant neoplasms. These terms typically apply to diseases characterized by unregulated cell division (or increased resistance to survival or apoptosis), and by the ability of these cells to invade other adjacent tissues (invasion), spread to other parts of the body where they are not normally located (metastasis), circulate through the bloodstream, and subsequently invade normal tissues elsewhere in the body. Tumors are classified as either benign or malignant depending on whether they can spread by invasion and metastasis; benign tumors are tumors that cannot spread by invasion or metastasis, i.e., grow only locally, while malignant tumors are tumors that can spread by invasion and metastasis. Biological processes known to be associated with cancer include angiogenesis, immune cell invasion, cell migration, and metastasis. Cancers typically share some of the following characteristics: persistent proliferative signaling, evasion of growth inhibitors, resistance to cell death, immortalization through replication, induction of angiogenesis, and activation of invasion and ultimately metastasis. Cancers can invade nearby parts of the body and can also spread to more distant parts of the body via the lymphatic system or bloodstream. Cancers are classified by the cell type that the tumor cells resemble, and thus are presumed to be the origin of the tumor.

[0295] Examples of cancer or tumors include, but are not limited to, tumors of the breast, heart, lungs, small intestine, colon, spleen, kidneys, bladder, head, neck, ovaries, prostate, brain, rectum, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, hepatobiliary tract, and liver. In particular, tumors / cancers include adenoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell carcinoma, glioblastoma, glioma, hemangioendothelioma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, hepatobiliary tract cancer, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, teratoma, acral lentiginous melanoma, actinic keratosis adenocarcinoma, adenoid cystic carcinoma, adenosarcoma, adenosquamous cell carcinoma, astrocytic tumors, Bartholin's gland carcinoma, basal cell carcinoma, bronchial gland carcinoma, carcinosarcoma, bile duct cancer, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymal sarcoma, Swing sarcoma, focal nodular hyperplasia, germ cell tumor, gulp The following may be selected from the group of tumors: cagonoma, hemangioblastoma, hemangioma, hepatic adenoma, hepatocellular adenoma, hepatocellular carcinoma, insulinoma, carcinoma in situ, squamous cell carcinoma in situ, invasive squamous cell carcinoma, large cell carcinoma, leiomyosarcoma, malignant melanoma, malignant mesothelioma, medullary epithelioma, mucoepidermoid carcinoma, neuroepithelial adenocarcinoma, nodular melanoma, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, renal cell carcinoma, serous carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vipoma, and Wilms' tumor.

[0296] In certain embodiments, the cancer is breast cancer. In preferred embodiments, the cancer is p95HER2-positive cancer.

[0297] The terms "p95HER2" and "HER2" have already been defined within the context of CAR in this invention, and those definitions apply equally to this diagnostic method.

[0298] "P95HER2-positive cancer" refers to cancer in which at least a portion of the cancer cells contain p95HER2, as determined by immunohistochemistry (IHC), Western blotting, or VeraTag® assay (Monogram Biosciences). In some embodiments, the cancer is determined to be p95HER2-positive by IHC. In some such embodiments, the cancer is determined to be p95HER2-positive using methods described in Sperinde et al., Clin. Canc. Res., 2010, 16(16):4226-4235, for example, using the anti-p95 antibody clone D9 in a VeraTag assay. In some embodiments, cancer is determined to be p95HER2 positive using a method described in U.S. Patent No. 8,389,227B2, for example, a method using antibodies produced by hybridoma cell lines deposited with the Deutschland Sammlung von Mikroorganisen and Zellen under accession number DSM ACC2904 or DSM ACC2980. In some embodiments, cancer is determined to be p95HER2 positive according to guidelines of the assay manufacturer or assay laboratory. p95HER2 refers to an aggregate of carboxy-terminal HER2 fragments, which in some embodiments may be divided into 95-100kDa and 100-115kDa fragments. See, for example, Arribas et al., Cancer Res., 2011, 71:1515-1519. In some embodiments, a p95HER2-positive cancer contains 100-115kDa fragments of HER2.

[0299] The terms “detection,” “diagnosing,” and “diagnosis,” or their derivatives, are used implicitly herein and refer to the identification of the presence or characteristics of a pathological condition. This refers to both the process of attempting to determine and / or identify a possible disease in a subject, i.e., the diagnostic procedure, and the opinion reached by this process, i.e., the diagnostic opinion. It can also be viewed as an attempt to classify the condition of an individual into distinct categories that enable medical decisions regarding treatment and prognosis to be made. As a person skilled in the art will understand, such a diagnosis may be preferable but not 100% correct in diagnosing a subject. However, in the context of this invention, the term requires that a statistically significant portion of a subject can be identified as having cancer. A person skilled in the art may determine whether a subject is statistically significant using different statistical evaluation tools known, such as determining confidence intervals, determining p-values, Student's test, Mann-Whitney, etc. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. The p-value is preferably 0.015, 0.001, 0.0005, or less.

[0300] Generally, the method involves obtaining a sample suspected of expressing the p95HER2 antigen under conditions effective in enabling the formation of an immune complex, and contacting the sample with an ScFv, antigen-binding domain, or antibody capable of selectively binding to or detecting the p95HER2 antigen.

[0301] The sample may be any sample suspected of containing the p95HER2 antigen, such as, for example, tissue sections or specimens, homogenized tissue extracts, cells, organelles, any isolated and / or purified form of any of the antigen-containing compositions described above, or any biological fluid including blood, serum, and plasma. In a preferred embodiment, the sample is a tumor sample. The sample is preferably a “tumor sample” which is derived from a patient’s tumor or a sample containing tumor cells derived from a patient’s tumor. Examples of tumor samples herein, but not limited to, include tumor biopsies, circulating tumor cells, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from or exhibiting tumor-like characteristics, and preserved tumor samples such as formalin-fixed paraffin-embedded tumor samples or frozen tumor samples.

[0302] Contacting the antibody of the present invention with a selected biological sample for a period sufficient to allow the formation of an immune complex under suitable and effective conditions generally involves simply adding the ScFv1, antigen-binding domain 1, or antibody of the present invention to the sample and incubating the mixture for a period long enough for the antibody to form an immune complex.

[0303] Effective conditions preferably involve diluting the sample and / or the ScFv1, antigen-binding domain 1, or antibody of the present invention with a solution such as BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween®. These added agents also tend to help reduce nonspecific background.

[0304] Furthermore, “suitable” or “appropriate” conditions mean that the incubation is at a temperature or for a period of time sufficient to allow for effective bonding. The incubation step may typically be at a temperature in the order of 25°C to 27°C for about 1 to 2 to 4 hours, or it may be at about 4°C overnight.

[0305] The amount of the formed complex can be determined in several ways. In a preferred embodiment, the antibody is labeled and binding is determined directly. For example, this can be done by binding the p95HER2 antigen protein to a solid support, adding a labeled ScFv, antigen-binding domain, or antibody (e.g., fluorescently labeled), washing away excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be used as are known in the art.

[0306] In general, the detection of immune complex formation is well known in the art and can be achieved by applying a number of approaches. These methods are generally based on the detection of labels or markers, such as their radioactive tags, fluorescent tags, biological tags, and enzyme tags. Naturally, as is known in the art, further advantages can be found by using secondary binding ligands such as a second antibody and / or biotin / avidin ligand binding configurations.

[0307] In certain embodiments, the ScFv1, antigen-binding domain 1, or antibody of the present invention is placed on a solid support.

[0308] ScFv, or other antigen-binding domains or other polypeptides such as antibodies, may be immobilized on various solid supports for use in assays. Solid phases that can be used to immobilize specific binding members include those developed and / or used as solid phases in solid-phase assays. Suitable solid phases include membrane filters, cellulosic paper, beads (including polymers, latex, and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multi-well plates. Assay strips can be prepared by coating ScFv, antigen-binding domains, or antibodies, or a combination thereof, arranged on a solid support. These strips could then be immersed in a test sample and rapidly processed through washing and detection steps to generate measurable signals, such as colored spots. ScFv, or other antigen-binding domains or other polypeptides such as antibodies, may be bound to specific zones of the assay device either by direct conjugate to the assay device surface or by indirect binding.

[0309] As those skilled in the art will understand, there is a wide range of conventional assays that can be used in the present invention using unlabeled ScFv1, antigen-binding domain 1, or antibody (primary antibody) and labeled antibody (secondary antibody) of the present invention. These techniques include Western blotting or immunoblotting, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich-ELISA), immunocytochemical and immunohistochemical techniques, flow cytometry, or multiplex detection techniques based on the use of protein microspheres, biochips, or microarrays containing ScFv1, antigen-binding domain 1, or antibody of the present invention. Other methods for detecting and quantifying the p95HER2 antigen using the antibody of the present invention include affinity chromatography techniques, ligand-binding assays, or lectin-binding assays.

[0310] Furthermore, unlabeled ScFv, antigen-binding domains, or antibodies must be detected with additional reagents, such as labeled secondary antibodies, which are then labeled. This is particularly useful for increasing the sensitivity of the detection method, as it allows the signal to be amplified.

[0311] In addition, antibody detection can also be carried out by detecting changes in the physical properties of the sample resulting from the binding of the antibody to its homologous antigen. These assays include determining transmission-related parameters in the sample, which are known in the art. As used herein, the term “transmission-related parameters” refers to parameters that indicate or correlate with the ratio of transmitted light to incident light in the sample, or parameters derived from them.

[0312] In one embodiment, transmission-related parameters are determined by turbidimetric or obscuration methods.

[0313] In another embodiment, the binding of the ScFv, antigen-binding domain, or antibody to its homologous antigen may be detected by surface plasmon resonance (SPR).

[0314] As used herein, SPR refers to the phenomenon in which the intensity of reflected light decreases sharply at a specific angle of incidence (i.e., resonance angle) when a laser beam is irradiated onto a thin metal film. SPR is a measurement method based on the phenomenon described above, which can analyze substances adsorbed on the surface of a thin metal film that acts as a sensor with high sensitivity. Subsequently, according to the present invention, for example, a target substance in a sample can be detected by immobilizing one or more ScFv, antigen-binding domains, or antibodies of the present invention on the surface of the aforementioned thin metal film, allowing the sample to pass through the surface of the thin metal film, and by detecting the difference in the amount of substance adsorbed on the surface of the thin metal film resulting from the binding of the ScFv, antigen-binding domain, or antibody to the target antigen before and after the sample passes through it.

[0315] In one embodiment, the presence of a bond, as measured by the above-mentioned related techniques or any other known in the art, indicates that the patient has cancer.

[0316] In another embodiment, the diagnostic method of the present invention includes comparing a level obtained in a subject under study conditions with a reference value, thereby indicating that an increased level of p95HER2 relative to the reference value indicates that the patient has cancer.

[0317] The term “increased” in relation to the level of p95HER2 refers to the expression of any level of p95HER2 detected using ScFv1, antigen-binding domain 1, or antibody according to the present invention in a sample that is lower than the reference value. Therefore, a p95HER2 expression level is considered decreased or below the reference value if it is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or lower than the reference value.

[0318] As used herein, the term “reference value” refers to a predetermined standard used as a criterion for evaluating values ​​or data obtained from samples collected from subjects. A reference value or reference level may be an absolute value, a relative value, a value with an upper or lower limit, a range of values, an average value, a median, a mean value, or a value compared to a specific control or baseline value. Reference values ​​may be based on values ​​from individual samples, for example, values ​​obtained from samples from subjects being tested, but at an earlier point in time. Reference values ​​may be based on a large number of samples, such as from a chronological age-matched population of subjects, or on a pool of samples that include or exclude the samples being tested. In one embodiment, the reference value corresponds to the level of p95HER2 expression determined in a healthy subject, thereby a healthy subject is understood to be a subject that does not exhibit proliferative disorder at the moment the level of p95HER2 expression is determined, and preferably does not exhibit a history of cancer.

[0319] In another embodiment, the reference value corresponds to the average or mean level of p95HER2 expression, determined from a pool of samples obtained from a group of patients who are clinically well-documented and disease-free, particularly cancer-free, and especially p95HER2-positive cancer-free. In such samples, the expression level may be determined, for example, by determining the mean expression level of the reference population. In determining the reference value, several characteristics of the sample type should be considered, such as the patient's age, sex, physical condition, or other characteristics. For example, the reference sample can be obtained from identical quantities of groups of at least 2, at least 10, and at least 100 to over 1000 individuals, such that the population is statistically significant.

[0320] As used herein, the terms “expression” or “expression level” refer to a measurable amount of protein or antigen. As will be understood by those skilled in the art, the expression level can be quantified by measuring the protein or antigen. In this case, the expression level of p95HER2 is measured by determining the amount of immune complex formed between the p95HER2 antigen and the ScFv1, antigen-binding domain 1, or antibody of the present invention, which can be done by several methods related to the above and known to those skilled in the art.

[0321] Pharmaceutical composition In a twelfth aspect, the present invention relates to a pharmaceutical composition comprising any of the host cells of the fourth aspect of the present invention, and / or ScFv1, antigen-binding domain 1, or antibody according to the fifth, sixth, and seventh aspects of the present invention, and at least one pharmaceutically acceptable excipient.

[0322] The term "pharmaceutical composition" refers to a form in which the biological activity of the active ingredient contained therein is effective, and which has unacceptable toxicity to the subject to which the composition is administered. It refers to a preparation that does not contain additional ingredients.

[0323] A "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to the target. Examples of pharmaceutically acceptable carriers, but not limited to these, include buffers, excipients, stabilizers, or preservatives.

[0324] In certain embodiments, the pharmaceutical composition comprises a host cell of the present invention, more specifically, an immune cell (e.g., a T, NK, or NKT cell) genetically engineered to express a CAR comprising any of the CARs of the present invention, namely ScFv1, antigen-binding domain 1, antigen-binding domain 2, or any combination thereof. In another embodiment, the pharmaceutical composition comprises ScFv1, antigen-binding domain 1, or an antibody. In yet another embodiment, the pharmaceutical composition comprises both a host cell and ScFv1, antigen-binding domain 1, or an antibody.

[0325] The pharmaceutical compositions and formulations described herein may be prepared by mixing an active ingredient of desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than approximately 10 residues). Polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoproteins like rHuPH20 (HYLENEX®, Baxter International, Inc.).

[0326] Treatment method The present invention provides a method for immunotherapy comprising administering a therapeutically effective amount of the ScFv1, antigen-binding domain 1, antibody, or immune cells of the present invention. In one embodiment, a medical disease or disorder is treated by transplantation of a population of immune cells that induces an immune response.

[0327] Accordingly, in the thirteenth aspect, the present invention relates to a host according to the fourth aspect of the present invention and / or any of the ScFv1, antigen-binding domain 1, or antibody according to the fifth, sixth, and seventh aspects of the present invention for use in pharmaceuticals.

[0328] In the final aspect, the present invention relates to any of the ScFv1, antigen-binding fragment 1, or antibody of the fifth, sixth, and seventh aspects of the present invention for use in host cells of the fourth aspect of the present invention and / or in methods for preventing or treating cancer.

[0329] In certain embodiments, the cancer is breast cancer. In preferred embodiments, the cancer is p95HER2 positive.

[0330] As used herein, “to treat,” “treatment,” “treatment,” or “improvement.” The term “treatment” refers to a therapeutic action whose purpose is to reverse, reduce, inhibit, delay, or halt the progression or severity of a condition associated with a disease or disorder. The term “treatment” includes reducing or alleviating at least one adverse effect or condition of a condition such as cancer, disease, or disorder. Treatment is usually “effective” if one or more symptoms or clinical markers are reduced; or if the progression of the disease is delayed or halted. That is, “treatment” includes not only improvement of symptoms or markers but also interruption of at least a condition that would indicate the expected progression or worsening of symptoms in the absence of treatment. Beneficial or desirable clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of the severity of the disease, a stable (i.e., non-worsening) state of the disease, or the disease itself. These include, but are not limited to, delayed or slowed progression, improvement or alleviation of the disease state, and remission (partial or total). The term “treatment” of a disease also includes providing relief from the symptoms or side effects of the disease (including symptomatic treatment). In some embodiments, treating cancer includes reducing tumor volume, reducing the number of cancer cells, inhibiting cancer metastasis, extending lifespan, reducing cancer cell proliferation, reducing cell survival, or reducing the cancerous state. This involves improvement of various physiological symptoms involved.

[0331] In certain embodiments of this disclosure, immune cells are delivered to an individual in need, such as an individual with cancer. The cells then enhance the individual's immune system and attack the respective cancer cells. In some cases, the individual is provided with one or more doses of immune cells. If the individual is provided with two or more doses of immune cells, the interval between doses should be sufficient to allow time for proliferation in the individual, and in certain embodiments, the interval between doses is one, two, three, four, five, six, seven days, or longer.

[0332] In some embodiments, the subject may be administered non-myeloid lymphocyte depletion chemotherapy prior to immunotherapy. Non-myeloid lymphocyte depletion chemotherapy may be any suitable such therapy, which may be administered by any suitable route. Non-myeloid lymphocyte depletion chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma and it may be metastatic. An exemplary route for the administration of cyclophosphamide and fludarabine is intravenous. Similarly, any suitable doses of cyclophosphamide and fludarabine may be administered. In a particular embodiment, about 60 mg / kg of cyclophosphamide is administered for 2 days, followed by about 25 mg / m2 of fludarabine for 5 days.

[0333] In certain embodiments, growth factors that promote the proliferation and activation of immune cells are administered to the target either simultaneously with or following the immune cells. The immune cell growth factor can be any suitable growth factor that promotes the proliferation and activation of immune cells. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7, and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2.

[0334] A therapeutically effective dose of immune cells may be administered via several routes, including parenteral administration, such as intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injection or infusion.

[0335] The immune cell population may be administered in a treatment regimen consistent with the disease, for example, as a single or multiple dose over one to several days to improve the disease state, or as a long-term, regular dose to inhibit disease progression and prevent disease recurrence. The exact dose used in the formulation also depends on the route of administration and the severity of the disease or disorder, and should be determined according to the practitioner's judgment and the individual patient's condition. The therapeutically effective number of immune cells depends on the subject being treated, the severity and type of distress, and the mode of administration. In some embodiments, the therapeutically effective number of immune cells is approximately 5 × 10¹⁰ per kg of body weight. 6 Cells ~ approximately 7.5 × 10 per kg of body weight 8 Cells, for example, about 2 × 10 per kg of body weight 7 Cells ~ approx. 5 x l0 8 Cells, or approximately 5 × 10 per kg of body weight 7 Cells ~ approx. 2×l0 8 Cellular variation is possible. The exact number of immune cells can be easily determined by those skilled in the art based on the subject's age, weight, sex, and physiological state. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0336] In certain embodiments, the compositions and methods of this embodiment include an immune cell population or ScFv in combination with at least one additional therapy. The additional therapy may be radiotherapy, surgery (e.g., mammary gland tumor removal and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of an adjuvant or neoadjuvant therapy.

[0337] In some embodiments, the additional therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, the additional therapy is the administration of side effect limiting agents (e.g., agents intended to reduce the occurrence and / or severity of therapeutic side effects, such as anti-nausea agents). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is a therapy targeting the PBK / AKT / mTOR pathway, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors, and / or chemopreventive agents. The additional therapy may be one or more chemotherapeutic agents known in the art.

[0338] The pharmaceutical compositions or immunotherapy of the present invention may be administered before, during, after, or in various combinations with additional cancer therapies such as immune checkpoint therapy. Administration may be simultaneous, at intervals ranging from minutes, days, or weeks. In some embodiments where immunotherapy is provided to the patient separately from additional therapeutic agents, it is generally ensured that a considerable period of time does not elapse between each delivery time so that the two compounds can still exert a combined effect beneficial to the patient. In such examples, it is intended that antibody therapy and anticancer therapy may be administered to the patient within approximately 12–24 hours or 72 hours of each other, more specifically, within approximately 6–12 hours of each other. In some situations, it may be desirable to significantly extend the duration of treatment, with days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsed between each administration.

[0339] Various combinations can be used. In the following example, the pharmaceutical composition or immunotherapy of the present invention is "A", and the anticancer therapy is "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0340] The administration of any compound or therapy of this embodiment to a patient follows a general protocol for the administration of such compound, taking into account the toxicity of the drug, if any. Therefore, in some embodiments, there is a step of monitoring for toxicity resulting from the combination therapy.

[0341] A wide variety of chemotherapeutic agents can be used in combination with the pharmaceutical compositions or immunotherapy of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These drugs or agents are classified, for example, by their mode of intracellular activity, whether and at what stage of the cell cycle they affect. Alternatively, drugs may be characterized based on their ability to induce chromosomal and mitotic abnormalities by directly crosslinking DNA, intercalating DNA, or affecting nucleic acid synthesis.

[0342] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocon, meteureopa, and uredopa; ethyleneimines and methylamelamamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomellamine; acetogenins (especially bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); briostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (synthetic Analogues, including KW-2189 and CB1-TM1); eleuterobin; pancratistatin; sarcodictiin; spongistatin; nitrogenous mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobenbitin, fenesterine, prednimustine, trophosphamide, and uracil mustard; nitroureas such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enegyoin antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omegal); dinemycin, including dinemycin A; bisphosphonates such as clodronate; esperamycin;Furthermore, neocardinostatin chromophores and related chromoprotein enegyoin antibiotic chromophores, acrasinomycin, actinomycin, ausralnisin, azaserin, bleomycin, kactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), Mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, and mitomycin C; mycophenolic acid, nogalar nisin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolbicin; metabolic resistance substances such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, pteropterin, and trimethrexate; fludarabine, 6-mercapto Purine analogs such as purines, thiamipurines, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as mitotane and trilostane; folic acid supplements such as floric acid; acegraston; aldofamide glycoside; aminolevulinic acid; enyluracil; amsac Phosphorus; Bestlovesil; Bisanthren; Edatraxate; Defofamine; Demecolsin; Diadicone; Elformitin; Erliptinium acetate; Epotilon; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidinin; Maytansinoids such as Maytansine and Ansamitosine; Mitoguazone; Mitoxanthrone; Mopidammol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxanthrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex; Lazoxane; Rhizoxin; Schizophyllan;Spirogermanium; tenuazonic acid; triadicone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, beracrine A, loridine A, and angiidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gasitosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16) Examples include ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; carboplatin, procarbazine, precomycin, gemcitabine, navelbine, farnesyl-protein tanspherase inhibitors, trans platinum, and any pharmaceutically acceptable salts, acids, or derivatives of the above. ***

[0343] The present invention is illustrated by the following examples, which are considered merely illustrative and do not limit the scope of the invention. [Examples]

[0344] Examples methodology Design and production of CAR vectors Vector plasmids encoding p95HER2 CARs (h32H2, 214D8, H1 214D8, H2 214D8, H3 214D8, 215C2, H1 215C2, H2 215C2) were synthesized and cloned into the pMSGV-1 retroviral vector (Genscript, Netherlands). Stocks of p95HER2 CARs, HER2 CARs, and empty (UTD) CAR retroviruses were then produced. Briefly, 0.7 μg of envelope plasmid (RD-114) and 1.5 μg of transfer plasmid (p95HER2, HER2, and empty CARs in pMSGV-1) were co-transfected into GP-293 cells (#631458, Clontech). After 2 and 3 days, the cell supernatant containing retroviral particles was collected and stored at -80C for future transduction.

[0345] Transduction and expansion of CAR T cells PBMCs were stimulated for 48 hours with 10 ng / µl α-CD3 (OKT3) (#16-0037-85, Thermo-Fisher) and 300 U / ml IL-2 (#703892-4, Novartis) before transduction. The cell supernatant containing retroviral particles was then thawed and centrifuged at 2000 g for 2 hours in a retronectin (#T100A, Takara) coated 6-well plate. Next, 2 × 10⁶ cells were extracted. 6 Stimulated PBMCs were added to the top layer and centrifuged at 400g for 10 minutes. After 5 days, CAR expression and cytotoxicity assays were performed. Untransduced T cells (UTDs) were transduced with empty CAR retroviruses.

[0346] CAR expression analysis 0.2 × 10 6CAR T cells were washed twice with 1x PBS and resuspended for 20 minutes in 1x PBS, 2.5 mM EDTA, 1% BSA, and 5% horse serum. The cells were then stained with 1 / 20 biotin anti-IgG (#115-065-072, Jackson ImmunoResearch) for 30 minutes and washed twice with 1x PBS. APC-streptavidin antibody (#405207, Biolegend) at 1 / 150 and 1 / 300 anti-CD3-PE (#300408, Biolegend) concentrations was added for 30 minutes. Zombie Aqua (#423101, Biolegend) was used as a viability marker at a 1:1000 dilution. CAR expression was measured using FACSCelesta (BD Bioscience) and analyzed using FlowJo software.

[0347] CAR T cell toxicity assay CFSE-labeled MCF10A p95HER2 / empty cells, shown E:CAR CAR T cells were co-cultured in 96-well flat-bottom plates at the T ratio. After 48 hours of incubation, the cell mixture was washed with 1x PBS and resuspended for 20 minutes in 1x PBS, 2.5 mM EDTA, 1% BSA, and 5% horse serum. The cells were then stained with zombie Aqua (#423101, Biolegend) at a 1:1000 dilution as a viability marker. CFSE-positive cells were counted using LSR Fortessa (BD Bioscience) and analyzed using FlowJo software.

[0348] InVivo Model NSG mouse, 3x10 6 MCF7p95HER2 / parent cells were injected orthotopic. Tumor volume was 300 mm². 3 When it reaches 3 × 10 6 CAR+ T cells were treated intravenously (iv) every 7–10 days with up to four doses. In the case of MCF7p95HER2 cells, mice were maintained in the presence of doxycycline (1 g / L) in drinking water.

[0349] result Example 1: Humanized 32H2 ​​p95HER2 CAR The anti-p95HER2 antibody 32H2 ​​is disclosed in a PCT application published as WO / 2010 / 000565. Initially, two versions of 32H2-derived p95HER2 CARs were generated using a single-stranded fragment variable (scFv) of the anti-p95HER2 antibody 32H2.

[0350] The two versions of the 32H2 ​​p95HER2 CAR differed in the arrangement of the lightly variable region (VL) and heavily variable region (VH) of the single-strand variable (scFv) of the 32H2 ​​antibody (Figure 2A). Both 32H2 ​​p95HER2 CARs contained a CD8 reader sequence (MALPVTALLLPLALLLHAARP SEQ ID NO: 147) at the beginning of the CAR sequence, a linker (TGSTSGSGKPGSGEGS SEQ ID NO: 29) between the variable regions, a CD8 hinge domain, a CD28 transmembrane domain and costimulatory domain, and a CD3 zeta domain. A trastuzumab-based CAR targeting full-length HER2 was used as a positive control (Figure 2A).

[0351] Neither of the generated 32H2 ​​p95HER2 CARs were detected on the cell surface (Figure 2B). Therefore, both 32H2 ​​p95HER2 CAR Ts were non-functional, as indicated by the lack of killing of MCF10A cells expressing p95HER2 (Figure 2C).

[0352] The scFv of 32H2 ​​was humanized, and two humanized versions, VH and VL, were obtained depending on the degree of humanization (Table 1). [Table 1] Table 1: Amino acid sequences of the heavily variable and lightly variable regions of different humanized 32H2 ​​versions: H1: humanized version 1; H2: humanized version 2.

[0353] Four versions of the humanized 32H2 ​​p95HER2 CAR were generated, differing in the order of the arrangement of the lightly variable region (VL) and heavily variable region (VH), as well as the humanized version used (H1 or H2). The four humanized 32H2 ​​p95HER2 CARs contained a CD8 reader sequence at the beginning of the CAR sequence, a linker between the variable regions, a CD8 hinge domain, a CD28 transmembrane domain and costimulatory domain, and a CD3 zeta domain (Figure 3A). The VL-VH H1 32H2 ​​p95HER2 CAR was expressed on the cell surface (Figures 3B, 4B), in contrast to the remaining humanized 32H2 ​​p95HER2 CAR versions (Figure 3B). Therefore, the VL-VH H1 32H2 ​​p95HER2 CAR was used in further experiments and named the humanized 32H2(h32H2)p95HER2 CAR.

[0354] Further experiments showed that h32H2 p95HER2 could be expressed on the cell surface at levels similar to trastuzumab-based CARs (Figure 4B). Furthermore, h32H2 p95HER2 CAR T cells co-cultured with MCF10A cells expressing p95HER2 induced specific cytotoxic effects (Figure 4C), but the efficacy was evident in a high proportion of target:CAR T cells. In contrast, h32H2 p95HER2 CAR T cells had no effect on MCF10A cells (Figure 4D), suggesting their specificity for p95HER2.

[0355] Example 2: 214D8 p95HER2 CAR The 214D8 p95HER2 CAR was generated from the scFv of the anti-p95HER2 antibody 214D8, the contents of which are disclosed in the U.S. Patent Application published as US2011 / 0135653, the entirety of which is incorporated herein by reference.

[0356] Two versions of the 214D8 p95HER2 CAR were developed with different arrangements of the lightly variable region (VL) and heavily variable region (VH) of the 214D8 antibody (Table 2, Figure 5A). Both 214D8 p95HER2 CARs contained the CD8 reader sequence, linker, CD8 hinge domain, CD28 transmembrane and costimulatory domains, and CD3 zeta domain.

[0357] Both 214D8 p95HER2 CARs were expressed on the cell surface, with the VL-VH 214D8 p95HER2 CAR being expressed at higher levels (Figure 5B). VL-VH 214D8 p95HER2 CAR T cells co-cultured with p95HER2-expressing MCF10A cells induced a high cytotoxic effect in a low proportion of target:CAR T cells (Figure 5C). [Table 2] Table 2. Amino acid sequences of the heavily variable and lightly variable regions of the 214D8 anti-p95HER2 antibody.

[0358] As shown in Table 3, humanized versions of the heavily variable and lightly variable regions of 214 anti-p95HER2 have also been obtained. [Table 3] Table 3. Amino acid sequences of the heavily variable and lightly variable regions of different humanized 214 anti-p95HER2 versions.

[0359] The humanized 214 anti-p95HER2 CAR version was expressed on the cell surface (Figure 6B), and at least the H1 214 and H2 214 humanized CAR versions induced high cytotoxicity even in a low proportion of target:CAR T cells (Figure 6D). In addition, as shown in Figure 6C, the use of the humanized ScFv version generates CAR T cells that are more specific to p95HER2 compared to the non-humanized version, due to reduced killing of cells expressing normal levels of HER2.

[0360] Example 3: 215C2 p95HER2 CAR The 215C2 p95HER2 CAR was generated from the ScFv of the anti-p95HER2 antibody 215C2.

[0361] Two versions of the 215C2 p95HER2 CAR were developed with different arrangements of the lightly variable region (VL) and heavily variable region (VH) of the 215C2 antibody (Table 4, Figure 7A). Both 215C2 p95HER2 CARs contained the CD8 reader sequence, linker, CD8 hinge domain, CD28 transmembrane and costimulatory domains, and CD3 zeta domain.

[0362] Both 215C2 p95HER2 CARs were expressed on the cell surface, and the VL-VH 215C2 p95HER2 CAR was expressed at a higher level (Figure 7B). Furthermore, VL-VH 215C2 was co-cultured with MCF10A cells expressing p95HER2. p95HER2 CAR T cells induced a high cytotoxic effect in a low proportion of target CAR T cells (Figure 7C). [Table 4] Table 4: Amino acid sequences of the heavily variable and lightly variable regions of the 215C2 anti-p95HER2 antibody

[0363] As shown in Table 5, humanized versions of the heavily variable and lightly variable regions of 215 anti-p95HER2 have also been obtained. [Table 5] Table 5. Amino acid sequences of the heavily variable and lightly variable regions of different humanized 215 anti-p95HER2 versions.

[0364] Humanized 215 anti-p95HER2 CAR versions H1 and H2 were expressed on the cell surface (Figure 8B), which also induced high cytotoxicity even in low proportion of target:CAR T cells (Figure 8C). In addition, as shown in Figure 8D, the use of the humanized ScFv version generates CAR T cells that are more specific to p95HER2 compared to the non-humanized version, due to reduced killing of cells expressing normal levels of HER2.

[0365] Example 4: Efficacy of m215-derived p95HER2 CAR T on the proliferation of p95HER2-positive tumors in vivo. MCF7p95HER2 cells were orthotopically transplanted into NSG mice. The tumor was approximately 300 mm. 3 When it reaches 3 × 10 6 The tumors were treated with m215-derived p95HER2 CAR+ T cells or UTD T cells. Complete remission of the tumor was achieved after three rounds of CAR+ T cell treatment (Figure 9A). Furthermore, circulating human CD3+ cells were detected after 35 days of treatment, suggesting adequate CAR T cell persistence.

[0366] Example 5: Efficacy of H1 214-derived p95HER2 CAR T cells on the proliferation of p95HER2-positive and p95HER2-negative tumors in vivo. NSG mice were orthotopically transplanted with MCF7p95HER2 cells or MCF7 cells. When the tumor reached approximately 300 mm3, the mice were treated with 3 × 10⁶ H1 214 p95HER2 CAR+ T cells or UTD T cells. Complete remission of the tumor was achieved after two rounds of H1 214 CAR T cell treatment when the tumor expressed p95HER2 (Figure 10A), suggesting high efficacy of H1 214-derived p95HER2 CAR T cells. In addition, when the tumor did not express p95HER2 but expressed normal levels of HER2, no effect on tumor growth was observed (Figure 10D), suggesting very high specificity of H1 214-derived CAR T cells to p95HER2. Furthermore, only when the tumor expressed p95HER2 (Figure 10B) did the circulating human CD3+ cell level increase 10 days after the second dose compared to the UTD group, but this was not observed in MCF7 tumors (Figure 10E), suggesting appropriate and specific CAR T expansion only in the presence of the congener antigen (Figure 10B). This result correlates with the level of human CD3+ in the tumor, as increased CD3+ cell infiltration was observed only in MCF7 p95HER2 tumors (Figure 10C) compared to the control group (UTD), but not in normal HER2-expressing MCF7 tumors (Figure 10F).

Claims

[Claim 1] The invention described in the specification.

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