Compositions and methods for immunotherapy of NPM1c-positive cancer

Abstract

We provide immunotherapy for acute myeloid leukemia (AML). [Solution] Provided are compounds (e.g., antibodies, their antigen-binding fragments, bispecific molecules, or chimeric antigen receptor polypeptides) that form a complex with major histocompatibility complex (MHC class I) proteins or bind to the neoepitope of mutant nucleophosmin (NPM1c) presented thereby, or cells expressing such compounds, and the use thereof in methods for treating or relieving one or more symptoms of cancer.

Description

[Technical Field]

[0001] Description of the government's implementation authority This invention was awarded by the National Institutes of Health (NIH) Grant No. CA1976. This was carried out with government support based on 05. The U.S. government has certain rights in this invention. do.

[0002] Cross-reference of related applications This application is based on U.S. Provisional Patent Application No. 62 / 987,612, filed on March 10, 2020. The interests of the specification are asserted. All of its contents are incorporated herein by reference. [Background technology]

[0003] Cell-based immunotherapy is being developed for the treatment of cancer. T cells, monocyte-derived cells ( For example, adoptive cell migration of macrophages, dendritic cells, and natural killer (NK) cells. Approaches using [specific technology / method] are being considered as cancer treatments (e.g., Andreesen, R. et al.). al (1990) Cancer Res 50:7450-7456; Ruggeri, L. et al (2002) Science 295:2097-21 See 00; Rezvani, K. (2019) Bone Marrow Transplantation 54:785-788). Specifically This is adoptive cell therapy (ACT), which involves administering T cells that have been activated / enlarged ex vivo to the patient. ) is one of the cancer treatments currently being tested. (Rosenberg et al. (2008) Nat Rev Cancer 8(4): 299; Dudley et al. (2002) Science 298(5594): 850; June et al. (2007 ) J Clin Invest 117(5): 1204; Stephan et al. (2007) Nat Med 13(12): 1440; Yee et al. al. (2002) Proc Natl Acad Sci USA 99(25): 16168). These approaches are patient It is extracted from, activated / enlarged ex vivo, and then used in tumors, such as metastatic tumors. This involves the use of autologous T cells that are reinjected to fight the disease. ACT T cell persistence, in v Strategies to increase IVO and enhance effector functions are aimed at increasing the frequency of objective effectiveness. It has been used for this purpose. (Rosenberg SA et al. (2008) Nat Rev Cancer 8(4): 299; June CH et al. (2007) J Clin Invest 117(5): 1204). One of the mechanisms that enhances the function of ACT T cells. One such method involves introducing chimeric receptors or costimulatory molecules into the cells themselves. This method involves genetic manipulation of the body (for example, Stephan et al. (2007) Nat Med 13(12): 1440; Morgan et al. (2006) Science 314(5796): 126; Gade et al. (2005) Cancer Res See 65(19): 9080).

[0004] Chimeric antigen receptor (CAR) T-cell therapy has emerged as one of the strategies for cancer treatment. Chimeric antigen receptors (CARs) are defined for an antigen (e.g., a ligand). Specificity is determined by immune effector cells (e.g., T cells, natural killer cells, or other immune cells). It is applied to cells, and when the cells recognize the antigen and bind to it, it activates the effector cells. These are genetically modified artificial transmembrane receptors. Typically, these chimeric receptors are The antigen specificity of monoclonal antibodies is determined for T cells, which are referred to in this technology as CAR T cells. Used to induce the development of manipulated chimeric antigen receptors on the surface of CAR T cells. Therefore, any target having surface expression of a specific antigen recognized by a chimeric receptor The ability to lyse cells is conferred to T cells.

[0005] However, current CA targets cell lineage-specific antigens or tumor-associated antigens (TAAs). R may be highly toxic due to low antigen expression in normal tissues (Coulie et al.) ., NAT REV CANCER 14: 135 (2014); Srivastava & Riddell, J IMMUNOL 200: 459 (2018 (See also). Furthermore, since TAA is not necessary for tumor cell survival, loss of TAA expression is , is a major cause of tumor resistance to CAR-T therapy (Srivastava & Riddell, See J IMMUNOL 200: 459 (2018). Neoantigens originate from tumor-specific gene mutations, and The formation and expression of CURR OPIN IMMUNOL are limited to malignant cells (Blankenstein et al., CURR OPIN IMMUNOL). 33 112 (2015); Schumacher et al. SCIENCE 348: 69 (2015); van der Lee et al., JC See LIN INVEST 129: 774 (2019). However, the vast majority of neoantigens are due to immunoediting. Patient-specific passenger mutations that may lead to loss of tumor immunity and subsequent evasion It is coded as such (see Verdegaal et al., NATURE 536: 91 (2016)). In addition, Current CARs are primarily designed to bind to antigens on the surface of target cells. In reality, most proteins derived from mutated genes are expressed within cells, and therefore, conventional It cannot be used as a target for CAR (see Uhlen et al., SCIENCE 347: 1260419 (2015)). see).

[0006] The application of cancer immunotherapy for acute myeloid leukemia (AML) is limited. It is one of the rapidly progressing hematopoietic malignancies, and is caused by malignant myeloid progenitor cells that have stopped differentiating in the bone marrow. Characterized by the accumulation of (van der Lee et al., J CLIN INVEST 129: 774 (2019); See Thomas et al., BLOOD 129: 1577 (2017). The current standard of treatment for AML is, Despite intensive chemotherapy and autologous or allogeneic hematopoietic stem cell transplantation (alloSCT), (Dombret & Gardin, BLOOD 127: 53 (2016); Dohner et al., N Engl J) See Med 373: 1136 (2015). Most patients achieve complete remission in response to standard treatment. However, relapses occur in about 50% of these patients (Ossenkoppele et al., HAEMA). See TOLOGICA 101 20 (2016). Relapse after intensive chemotherapy or alloSCT. Patients with refractory AML usually have a very poor prognosis (van der Lee et al., J CLIN I (See NVEST 129: 774 (2019)), therefore, new and effective treatments for these patients are toxic. There is a great need to develop treatments that minimize side effects. [Overview of the Initiative]

[0007] In some embodiments, this disclosure relates to Class I major histocompatibility complex (MHC class I) proteins. An antibody that specifically binds to an antigen containing an NPM1c neoepitope that has formed a complex with the substance, It provides the antigen-binding fragment.

[0008] In some embodiments, the antibody or its antigen-binding fragment is (a) a single MHC class I protein German, and / or (b) control peptides that form a complex with MHC class I proteins It does not bind, or binds substantially, and optionally, the control peptide is NY-ESO This is a -1 epitope or influenza virus M1 epitope.

[0009] In some embodiments, the antibody or its antigen-binding fragment is (a) a single MHC class I protein (b) The control peptide that forms a complex with MHC class I proteins is bound to (b) the control peptide. If not present or substantially unbound, the control peptide is either NY-ESO-1 epidithenic peptide (optional). It is a tope or influenza virus M1 epitope.

[0010] In some embodiments, the antibody, or its antigen-binding fragment, is (a) an MHC class I protein. (b) A control peptide alone, or a control peptide that has formed a complex with an MHC class I protein, at any choice. The control peptide is either the NY-ESO-1 epitope or the influenza virus M1 epitope. It is a pe and / or does not bind to (c)NPM1c neoepitope alone, or They are not substantially combined.

[0011] In some embodiments, the antibody, or its antigen-binding fragment, is (a) an MHC class I protein. (b) A control peptide alone, or a control peptide that has formed a complex with an MHC class I protein, at any choice. The control peptide is either the NY-ESO-1 epitope or the influenza virus M1 epitope. (c)NPM1c neoepitope does not bind to the neoepitope alone, or does not bind to it substantially. They do not bind to each other.

[0012] In any of the above embodiments or related embodiments, the NPM1c neoepitope is A The formula contains a mino acid sequence X1X2X3X4X5X6X7X8X9, where X1 is A, V, L or X1 is selected from I, X2 is selected from A, T, S, V, L, I, M or Q, and X3 is Q Or selected from N, X4 is selected from D or E, X5 is L, I, V, M, A or X6 is selected from F, X6 is selected from C, S, or A, X7 is L, I, V, M, A, Alternatively, X8 is selected from F, and X9 is selected from A, V, L or I, and X9 is selected from L, I, V, M Or selected from A. In some embodiments, the NPM1c neoepitope is an amino acid sequence The formula includes X1, X2, X3, X4, X5, X6, X7, X8, and X9, where X1 is selected from A or V. X2 is selected from V, I, or L, X3 is selected from Q or N, and X4 is D or Selected from E, X5 is selected from L or I, X6 is selected from C or S, X7 X8 is selected from V, L or I, X8 is selected from A or V, X9 is V, I or Selected from L. In some embodiments, the NPM1c neoepitope is the amino acid sequence X1X The formula includes 2X3X4X5X6X7X8X9, where X1 is A and X2 is V, I, or Selected from L, X3 is Q, X4 is D, X5 is L, and X6 is C. X7 is L, X8 is A, and X9 is selected from V, I, or L. Some embodiments So, the NPM1c neoepitope is 10, 15, 20, 30, 40, 50 or 100 It is located within a peptide with an amino acid residue length.

[0013] In any of the above embodiments or related embodiments, the NPM1c neoepitope is A Select from IQDLCLAV (sequence number 1) or AIQDLCVAV (sequence number 71) It contains an amino acid sequence. In some embodiments, the NPM1c neoepitope is CLAVEE VSL (sequence code 72), VEEVSLRK (sequence code 73), AVEEVSLR (array Number 74), AVEEVSLRK (Sequence ID 75), CLAVEEVSLRK (Sequence ID Includes an amino acid sequence selected from 76). In some embodiments, NPM1c neoepitope It contains the amino acid sequence AIQDLCLAV (SEQ ID NO: 1).

[0014] In any of the above embodiments or related embodiments, the NPM1c neoepitope is 7 The length is 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues.

[0015] In any of the above embodiments or related embodiments, the MHC class I protein is H Whether it is an LA-A*02 protein or determined by the HLA-A*02 allele group In some embodiments, MHC class I proteins are HLA-A*02:01. Coded by Rel.

[0016] In any of the aforementioned embodiments or related embodiments, the Disclosure shall (i) Heavy chain variable region (VH) complementarity determination region (CDR) 1, VH CDR2 and VH A VH including CDR3, and the VH CDR1, VH CDR2 and VH CD R3 is a VH CDR having the amino acid sequence of SEQ ID NO: 5, and the CDR is IMGT As defined, VH; and / or (ii) Light chain variable region (VL) complementarity determination region (CDR) 1, VL CDR 2 and V A VL including L CDR3, wherein VL CDR1 and VL CDR2 and VL C DR3 is a VL CDR having the amino acid sequence of SEQ ID NO: 3, and the CDR is in IMGT. Therefore, as defined, VL The present invention provides an antibody containing an antigen-binding fragment thereof.

[0017] In any of the above-described embodiments or related embodiments, the Disclosure relates to the heavy chain variable region (VH) VH including complementarity determination region (CDR) 1, VH CDR2 and VH CDR3 VH CDR1 has the amino acid sequence GFTFSSYA (SEQ ID NO: 9), VH CDR 2 has the amino acid sequence ISGSGGST (SEQ ID NO: 10), and VH CDR3 is an amino acid An antibody containing VH having the sequence ARLGYPTTTLLPFDY (SEQ ID NO: 11), It provides the antigen-binding fragment.

[0018] In any of the above-described embodiments or related embodiments, the Disclosure relates to the light chain variable region (VL). A VL that includes complementarity determination region (CDR) 1, VL CDR2, and VL CDR3. VL CDR1 has the amino acid sequence QSISSY (SEQ ID NO: 6), and VL CD2 has It has the amino acid sequence AAS (SEQ ID NO: 7), and VL CD3 has the amino acid sequence QQSYSTPL This provides an antibody having T (Sequence ID 8) and further containing VL, or an antigen-binding fragment thereof. .

[0019] In any of the above-described embodiments or related embodiments, the Disclosure relates to the heavy chain variable region (VH) and an antibody or antigen-binding fragment thereof comprising a light chain variable region (VL), wherein VH is distributed The amino acid sequence of column number 5 is at least 90% identical, or at least 95% identical. The amino acid sequence is included, and / or VL is at least 90 amino acids from the amino acid sequence of SEQ ID NO: 3. An antibody or its antigen containing an amino acid sequence that is 100% identical, or at least 95% identical. Provides a binding fragment.

[0020] In any of the above-described embodiments or related embodiments, the Disclosure relates to the heavy chain variable region (VH) An antibody or antigen-binding fragment thereof, comprising a light chain variable region (VL), wherein VH is a sequence It contains the amino acid sequence of number 5, and / or VL contains the amino acid sequence of SEQ ID NO: 3. The present invention provides an antibody or an antigen-binding fragment thereof.

[0021] In any of the above-described or related embodiments, the Disclosure refers to human antibodies, humanized antibodies. Alternatively, the present invention provides an antibody that is a chimeric antibody, or an antigen-binding fragment thereof.

[0022] In any of the above-described or related embodiments, the Disclosure relates to single-chain Fv(scFv) Fv fragment, Fab fragment, Fab' fragment, F(ab')2 fragment, or single-chain antibody molecule The present invention provides an antibody or an antigen-binding fragment thereof.

[0023] In any of the above-described embodiments or related embodiments, the Disclosure relates to an antibody that is an scFv, The present invention provides either an antigen-binding fragment thereof. In some embodiments, the scFv is a human scFv. In some embodiments, scFv includes a linker. In some embodiments, the linker is peptidine It is a car. In some embodiments, the peptide linker is a Gly-Ser linker. In this embodiment, the Gly-Ser linker is (Gly4Ser)(SEQ ID NO: 58), (Gl (Gly4Ser)2 (Sequence ID 59), (Gly4Ser)3 (Sequence ID 60), and (G Selected from the group consisting of ly4Ser)4 (Sequence No. 61). In some embodiments, Gly - The Ser linker contains the amino acid sequence SGSSGGSSSG (SEQ ID NO: 4).

[0024] In any of the above embodiments or related embodiments, scFv is the amino acid of SEQ ID NO: 2 The acid sequence is at least 80% identical, at least 85% identical, at least 90% identical, Or, having at least 95% identical amino acid sequences, and optionally, scFv is (a) heavy Chain Variable Region (VH) Complementarity Determination Region (CDR) 1, VH CDR2, and VH CDR3 VH containing the amino acid sequence GFTFSSYA (SEQ ID NO: 9) VH CDR2 has the amino acid sequence ISGSGGST (SEQ ID NO: 10), and VH CDR3 has the amino acid sequence ARLGYPTTTLLPFDY (SEQ ID NO: 11), V H; and / or (b) Light chain variable region (VL) complementarity determination region (CDR) 1, VL C VL containing DR2 and VL CDR3, where VL CDR1 is the amino acid sequence QSI SSY (SEQ ID NO: 6) is present, and VL CD2 has the amino acid sequence AAS (SEQ ID NO: 7). VL CD3 has the amino acid sequence QQSYSTPLT (SEQ ID NO: 8) and contains VL. .

[0025] In the above embodiment or any related embodiment, scFv is the amino acid of SEQ ID NO: 2 It has an array.

[0026] In any of the above embodiments or related embodiments, the Disclosure relates to an antibody which is an antibody, and It provides the antigen-binding fragment. In some embodiments, the antibody is IgG1, IgG2, IgG 3. IgG4, IgM, IgA1, IgA2, IgD, and IgE antibody isotypes Selected from the following group. In some embodiments, the antibody is IgG1 isotype or IgG It is an isotype. In some embodiments, the antibody is a single heavy chain constant region of wild-type IgG. or contains the wild-type IgG quadruple chain constant region. In some embodiments, the antibody contains the mutant IgG monocoque. Includes a constant region or a variant IgG quadruplex constant region.

[0027] In any of the above embodiments or related embodiments, the antibody is a mutant IgG quadrilateral constant region The variant IgG quadrilateral constant region includes one of the following substitutions: E S228P, L235E, L235A, or a combination thereof, assigned by U-numbering.

[0028] In any of the above embodiments or related embodiments, the antibody has at least one mutation Includes FC domains.

[0029] In any of the above-described embodiments or related embodiments, the antigen is located on the surface of the cancer cell. In some cases, the cancer is acute myeloid leukemia (AML).

[0030] In any of the above embodiments or related embodiments, the antibody, or its antigen-binding fragment, NPM that forms complexes with Class I major histocompatibility complex (MHC class I) proteins Antigens containing 1c neoepitopes: 100 nM or less, 50 nM or less , 20 nM or less, 10 nM or less, 0.5 nM to 100 nM, Alternatively, they bind with an equilibrium dissociation constant (Kd) ranging from 1 nM to 15 nM.

[0031] In any of the above-described or related embodiments, the Disclosure refers to immune effector cells. A bispecific antibody, or its antigen-binding fragment, that further specifically binds to the second antigen mentioned above. The present invention provides an antibody or an antigen-binding fragment thereof. In some embodiments, effector cells are These are T cells, natural killer cells, or macrophages. In some embodiments, a second antibody The original is CD3. In some embodiments, CD3 is human CD3 expressed on T cells. In some embodiments, the second antigen is NKp46. In some embodiments, NKp46 is This is human NKp46 expressed on NK cells. In some embodiments, the second antigen is CD16. A. In some embodiments, CD16A is human CD16A expressed on NK cells. In some embodiments, the second antigen is CD40, CD47, 4-1BB, TGF-β, L AG-3, PD-1, TIM-3, CTLA-4, OX40, NKp30, NKG2A, It is either NKG2D or DNAM-1.

[0032] In any of the above-described or related embodiments, the Disclosure relates to purified antibodies, and It provides the antigen-binding fragment.

[0033] In some aspects, this disclosure relates to isolated nucleic acids, wherein the nucleic acids are described herein. The present invention provides nucleic acids containing a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof.

[0034] In any of the above embodiments or related embodiments, the isolated nucleic acid is SEQ ID NO: 12 It contains the nucleotide sequence. In some embodiments, the isolated nucleic acid is shown in SEQ ID NO: 12 The nucleotide sequence and at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, It contains nucleotide sequences with 96%, 97%, 98%, or 99% identity.

[0035] In some embodiments, this disclosure provides an expression vector comprising nucleic acids described herein. In some aspects, this disclosure is shaped by an expression vector containing nucleic acids described herein. We provide converted cells.

[0036] In some embodiments, this disclosure makes an antibody or its antigen-binding fragment described herein. A method for producing a trait by an expression vector containing nucleic acids as described herein. The converted cells are maintained under conditions that allow for the expression of antibodies or their antigen-binding fragments. The present invention provides a method comprising the steps of binding an antibody or its antigen. In some embodiments, the method involves binding an antibody or its antigen. The process further includes a step of purifying the fragments.

[0037] In some embodiments, this disclosure relates to the therapeutic effects of antibodies or antigen-binding fragments thereof as described herein. The present invention provides a pharmaceutical composition comprising a therapeutically effective amount and a pharmaceutically acceptable carrier.

[0038] In some embodiments, this disclosure relates to intracellular domains, transmembrane domains and extracellular binding domains. A chimeric antigen receptor (CAR) polypeptide containing a chromosome, wherein the extracellular binding domain is a chromosome. NPM1c complexed with major histocompatibility complex I (MHC class I) proteins. This invention provides a CAR polypeptide that specifically binds to antigens containing neoepitopes.

[0039] In some embodiments, this disclosure relates to intracellular domains, transmembrane domains and extracellular binding domains. A chimeric antigen receptor (CAR) polypeptide containing the extracellular binding domain, The specification provides a CAR polypeptide comprising the antibody described herein, or its antigen-binding fragment. ru.

[0040] In any of the above embodiments or related embodiments, the transmembrane domain is CD3-ze Transmembrane dendrocytes (T, CD8, CD28, NKG2D, CD16, NKp44, or NKp46) Includes the main. In some embodiments, the intracellular domain is CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, 2B4, DAP10, CD137 and one or more co-stimulatory molecules selected from the group consisting of DAP12 Includes a numerical co-stimulation domain.

[0041] In any of the above-described or related embodiments, the Disclosure relates to the intracellular domain being CD It contains a 3-zeta signaling domain and a 4-1BB costimulatory domain, and transmembrane domain The molecule contains a CD8 transmembrane domain, and the CAR polypeptide further contains a CD8 hinge region. , provides CAR polypeptides.

[0042] In any of the above-described or related embodiments, the Disclosure is a CAR polypeptide. The intracellular domain contains the amino acid sequence shown in Sequence ID No. 27, which is CD3-zetase. The 4-1BB cosectose includes the Gunal signaling domain and the amino acid sequence shown in SEQ ID NO: 26. Including the stimulating domain, the CAR polypeptide contains the CD8 transmembrane domain and the CD8 hinge region. The CD8 transmembrane domain and CD8 hinge region are included in the region shown in Sequence ID No. 25. The extracellular binding domain contains an antibody, or its antigen-binding fragment, and the sequence A CAR polypeptide containing a leading sequence including the amino acid sequence shown in number 23 provide.

[0043] In any of the above embodiments or related embodiments, the extracellular binding domain contains an antibody The antigen-binding fragment is the amino acid sequence shown in SEQ ID NO: 24, or the sequence of SEQ ID NO: 24 At least 70% identical to the amino acid sequence, at least 75% identical, at least 80% identical One, at least 85% identical, at least 90% identical, or at least 95% identical This is an scFv containing a single amino acid sequence.

[0044] In any of the above embodiments or related embodiments, the intracellular domain is self-cleaving Further containing peptide sequences and cytokines, the self-cleaving peptide cleaves the cytokine In some embodiments, cytokines are released. 3. IL-15, TNF-α, IFN-γ, or CCL19.

[0045] In any of the above-mentioned embodiments or related embodiments, the Disclosure is as shown in Sequence ID No. 22. The amino acid sequence, or at least 70% identical to the amino acid sequence of SEQ ID NO: 22, Both are 75% identical, at least 80% identical, at least 85% identical, at least 90% We propose CAR polypeptides containing 100% identical, or at least 95% identical, amino acid sequences. To provide.

[0046] In some aspects, this disclosure may include single CAR polypeptides described herein. Provides isolated nucleic acids. In some embodiments, the isolated nucleic acids are nucleos of SEQ ID NO: 30. The nucleotide sequence, or the nucleotide sequence of SEQ ID NO: 30, is at least 70% identical, at 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, also or include nucleotide sequences that are at least 95% identical. In some embodiments, the disclosure , an expression vector comprising isolated nucleic acids encoding the CAR polypeptide described herein It is a vector, and the expression vector is either a viral expression vector or a non-viral expression vector. This disclosure provides an expression vector. In some embodiments, this disclosure provides an expression vector as described herein. An expression vector comprising isolated nucleic acids encoding a lipeptide, wherein the expression vector is It is a virus expression vector, and the virus expression vector is a lentiviral expression vector. We provide an expression vector.

[0047] In some aspects, this disclosure may include single CAR polypeptides described herein. The present invention provides cells transformed with an expression vector containing detached nucleic acids. In some embodiments, This disclosure provides cells expressing the CAR polypeptides described herein. In this particular case, the cells are immune effector cells, and the expression of CAR polypeptides is immune Effector cells are combined with Class I major histocompatibility complex (MHC class I) proteins. This targets cancer cells that express antigens containing the NPM1c neoepitope, which has formed a complete body. In some embodiments, MHC class I proteins are HLA-A*2 proteins, or encoded by the HLA-A*02 allele group. In some embodiments, immunology Injector cells do not substantially target cancer cells expressing wild-type NPM1, and / or However, it does not induce its elimination. In some cases, immune effector cells are wild-type NPM1 It does not substantially target cancer cells that express [the specified gene]. In some embodiments, immune effector cells Furthermore, it does not substantially induce the killing of cancer cells expressing wild-type NPM1. In some embodiments, The cells are T cells. In some forms, T cells are human CD8 + This is a T cell. Then the cell is a natural killer (NK) cell. In some embodiments, the cell is a macrophage. In some cases, cancer cells are acute myeloid leukemia (AML) cells.

[0048] In some aspects, this disclosure may include single CAR polypeptides described herein. Cells transformed with an expression vector containing detached nucleic acids and pharmaceutically acceptable carrier The present invention provides a pharmaceutical composition comprising the body. In some embodiments, this disclosure is described herein as C The present invention provides a pharmaceutical composition comprising cells expressing an AR polypeptide and a pharmaceutically acceptable carrier. do.

[0049] In some embodiments, this disclosure relates to cells expressing the CAR polypeptides described herein. A method for producing (i) cells from target peripheral blood mononuclear cells (PMBCs) (ii) optionally, an anti-CD3 antibody, or its antigen-binding fragment and / Alternatively, a step of activating cells with an anti-CD28 antibody or its antigen-binding fragment, iii) Derived from isolated nucleic acids encoding the CAR polypeptide described herein. The steps are: (iv) Transduction of cells using the vector, and (iv) Expression of CAR polypeptide. (v) a step of isolating the cells, and optionally a step of growing the isolated cells. A method including steps is provided.

[0050] In some embodiments, this disclosure relates to cells expressing the CAR polypeptides described herein. A method for producing (i) pluripotent stem cells (iPSCs) and immunofluid (ii) a step of differentiating into dermatomorph cells, and (ii) a CAR polypeptide described herein. An expression vector containing an isolated nucleic acid encoding the immunoeffector cell is used to induce plasma induction in immune effector cells. (iii) Isolate immune effector cells expressing CAR polypeptides. (iv) optionally, to amplify isolated immunoeffector cells. A method is provided that includes the step of [doing something].

[0051] In any of the above-described embodiments or related embodiments, immune effector cells are NK cells. In some embodiments, the immune effector cells are macrophages. Therefore, immune effector cells are T cells.

[0052] In certain aspects, this disclosure relates to a method for treating cancer in a person in need of cancer treatment. The cell surface of cells including cancer cells is a Class I major histocompatibility complex (MHC Class I ) Presenting the NPM1c neoepitope that has formed a complex with the protein, as described herein. Antibodies, or antigen-binding fragments thereof, cells as described herein, or as described herein A method comprising the step of administering the listed pharmaceutical composition to a subject in an amount sufficient to treat cancer. Provides the law. In some aspects, cancer is acute myeloid leukemia (AML). In some aspects So, what are the methods of treating cancer? How can we reduce the amount of cancer, or how can we improve the patient's survival time? This is a method to extend it.

[0053] In certain aspects, this disclosure is relevant to subjects requiring treatment for acute myeloid leukemia (AML). A method for treating AML, comprising an antibody or antigen binding thereof as described herein. Fragments, cells as described herein, or pharmaceutical compositions as described herein, are used to treat AML The present invention provides a method comprising the step of administering to a subject a sufficient amount to treat the condition. In some embodiments, AML can be relapsed AML or refractory AML.

[0054] In some aspects, this disclosure may help prevent AML relapse in subjects who are in remission from AML. A method comprising an antibody or antigen-binding fragment thereof as described herein, as described herein The step includes administering the cells described or the pharmaceutical composition described herein. To provide a method.

[0055] In any of the above-described or related embodiments, the Disclosure refers to the following prior to the administration step. , whether the subject expresses NPM1c or not, or whether the subject has NPM1c in the NPM1 gene The system detects whether or not a mutation is present, and determines whether the subject expresses NPM1c or has an NPM1c mutation. The present invention provides a method that includes a step of proceeding to the administration step if necessary.

[0056] In any of the above-described embodiments or related embodiments, the Disclosure relates to administration being intravenous, intrathecal, or intrathecal. The invention provides a method that is intraosseous or intraspinal.

[0057] In any of the above embodiments or related embodiments, the method includes one or more additional The further step includes administering a therapeutic agent or procedure. In some embodiments, the additional therapeutic agent is It is an inhibitor of an immune checkpoint molecule, and is selected at will, and the immune checkpoint molecule is T The inhibitor is either IM-3, PD-1, PD-L1, or CTLA-4, and is selected at will; the inhibitor is an antibody. That is the case.

[0058] In some aspects, this disclosure relates to the manufacture of a pharmaceutical product for treating cancer in the subject, Antibodies as described herein, or antigen-binding fragments thereof, CARPs as described herein Use of lipeptides, cells as described herein, or pharmaceutical compositions as described herein The cell surface of cells including cancer cells is a Class I major histocompatibility complex (MHC Class I ) Present the NPM1c neoepitope that has formed a complex with the protein; optionally, use However, it is provided for use in combination with one or more additional therapeutic agents or procedures. do.

[0059] In any of the above-described embodiments or related embodiments, the subject is a human being.

[0060] In some embodiments, this disclosure includes (i) antibodies or antigens described herein. Fragments, CAR polypeptides as described herein, cells as described herein, or this (ii) Pharmaceutical compositions as described in the specification; (ii) optionally one or more additional therapeutic agents and (iii) one or more instructions for use in the treatment of cancer in the subject We provide a kit that includes several containers.

[0061] In another embodiment, MHC (e.g., MHC class I) proteins (e.g., HLA-A2) ) is particularly relevant to antigens containing neoepitopes that form complexes with (or are presented by) them. Antibodies or antigen-binding fragments that bind heteroto are described herein. In certain embodiments, MHC (e.g., MHC class I) proteins (e.g., HLA- Antigens containing neoepitopes that have formed a complex with (or are presented by) A2) An antibody or its antigen-binding fragment that specifically binds to the antigen is, for example, (i) an antibody that binds to the antigen. The clones are displayed in a yeast surface display antibody (e.g., scFv) library or fur. The step of isolation using a didisplay antibody (e.g., scFv) library, (ii) Yeast surface display antibody (e.g., scFv) library or fur Using a didisplay antibody (e.g., scFv) library, multiple antibody clones are generated. Multiple positive selections (2, 3, 4 or more times) (yeast or fylliols that bind to the antigen) Select a clone of the purge and do it multiple times (2, 3, 4 or more times) Negative selection (e.g., MHC protein alone and / or control peptide (i.e., negative selection) Yeast that binds to MHC proteins (which form a complex with peptides different from the oepitopes) By subjecting the phage clones to a process that selectively removes them, the antigen is specifically bound. The antibody clones are generated by the step of selecting the matching antibody clones; the selected antibody clones are It binds to antigens and forms complexes with MHC proteins alone and / or control peptides. It does not bind to MHC proteins, or does not bind substantially. In certain aspects of this embodiment In this embodiment, the antigen is a dimeric neoepitope-MHC complex. In certain embodiments, the neoepitope-MHC complex is NPM1c:HLA-A2 ru. [Brief explanation of the drawing]

[0062] [Figure 1-1]Figures 1A-1D illustrate the isolation of AIQ-HLA-A2 complex-specific human scFv by yeast surface display. Figure 1A shows a schematic of the epitope peptide-HLA-A2 complex, scFv displayed on the yeast surface, and the binding of the peptide-HLA-A2 complex to scFv on the yeast cell surface. The schematic diagram was adapted from Chao et al., NAT PROTOC 1: 755 (2006). [Figure 1-2] Figures 1A–1D show the isolation of human scFv specific to the AIQ-HLA-A2 complex by yeast surface display. Figures 1B-1 and 1B-2 show the strategies and steps used to isolate yeast cells displaying scFv that specifically recognize the AIQ-HLA-A2 complex. The rounds of selection are shown on the left (rounds 1–5 in Figure 1B-1, and rounds 6–9 in Figure 1B-2). "Antigen" indicates the peptide-HLA-A2 complex or HLA-A2 alone used for positive or negative selection. In the first two rounds of selection, yeast cells were selected by magnetic cell sorting (MACS). In the remaining rounds of selection, yeast cells were sorted by flow cytometry based on staining with FITC-labeled anti-c-Myc antibody and PE-labeled anti-mouse IgG or APC-labeled streptavidin. Gating for sorted cells is shown. FACS plots are labeled #1 through #7. [Figure 1-3]Figures 1A–1D show the isolation of human scFv specific to the AIQ-HLA-A2 complex by yeast surface display. Figures 1B-1 and 1B-2 show the strategies and steps used to isolate yeast cells displaying scFv that specifically recognize the AIQ-HLA-A2 complex. The rounds of selection are shown on the left (rounds 1–5 in Figure 1B-1, and rounds 6–9 in Figure 1B-2). "Antigen" indicates the peptide-HLA-A2 complex or HLA-A2 alone used for positive or negative selection. In the first two rounds of selection, yeast cells were selected by magnetic cell sorting (MACS). In the remaining rounds of selection, yeast cells were sorted by flow cytometry based on staining with FITC-labeled anti-c-Myc antibody and PE-labeled anti-mouse IgG or APC-labeled streptavidin. Gating for sorted cells is shown. FACS plots are labeled #1 through #7. [Figure 1-4] Figures 1A–1D show the isolation of human scFv specific to the AIQ-HLA-A2 complex by yeast surface display. Figures 1C-1 and 1C-2 show flow cytometry data (FACS) for viable cells. Selected yeast cells from rounds 4 through 9 were augmented, then stained with FITC-labeled anti-c-Myc antibody and biotin-labeled HLA-A2, GIL-HLA-A2, SLL-HLA-A2, or AIQ-HLA-A2, followed by APC-labeled streptavidin, and gated by flow cytometry for viable cells (DAPI-negative). Data for rounds 4 and 5 are shown in Figure 1C-1, and data for rounds 6–9 are shown in Figure 1C-2. FACS plots were labeled from #1 to #29. [Figure 1-5]Figures 1A–1D show the isolation of human scFv specific to the AIQ-HLA-A2 complex by yeast surface display. Figures 1C-1 and 1C-2 show flow cytometry data (FACS) for viable cells. Selected yeast cells from rounds 4 through 9 were augmented, then stained with FITC-labeled anti-c-Myc antibody and biotin-labeled HLA-A2, GIL-HLA-A2, SLL-HLA-A2, or AIQ-HLA-A2, followed by APC-labeled streptavidin, and gated by flow cytometry for viable cells (DAPI-negative). Data for rounds 4 and 5 are shown in Figure 1C-1, and data for rounds 6–9 are shown in Figure 1C-2. FACS plots were labeled from #1 to #29. [Figure 1-6] Figures 1A–1D show the isolation of human scFv specific to the AIQ-HLA-A2 complex by yeast surface display. Figure 1D shows flow cytometry data. Yeast cells expressing YG1 or YG2 clones were stained and analyzed as shown in Figure 1C. The percentages in Figures 1B, 1C, and 1D represent the percentage of cells within the gated region. [Figure 2-1] Figures 2A-2E show the specific and high-affinity binding of YG1 scFv to the AIQ-HLA-A2 complex on AML cells. Figure 2A shows a schematic of a switchable yeast display / secretion vector for expressing the scFv-Fc fusion protein. In this switchable system, scFv-Fc can be secreted or displayed on yeast cells depending on whether or not OmeY is added to the culture (Van Deventer et al., PROTEIN ENG DES SEL 28: 317 (2015)). Figure 2B shows the SDS-PAGE analysis of the purified YG1 scFv-Fc protein. Lane 1: Protein ladder, Lane 2: Unreduced scFv-Fc protein (1 μg), Lane 3: Reduced scFv-Fc protein (1 μg). The gel was stained with Coomassie blue. [Figure 2-2]Figures 2A-2E show the specific and high-affinity binding of YG1 scFv to the AIQ-HLA-A2 complex on AML cells. Figure 2C shows flow cytometry data of HLA-A2 expression in OCI-AML3, T2, GMB, PC-3, and OCI-AML2 cells. Darkly shaded histograms are stained with anti-HLA-A2, and lightly shaded histograms are stained with isotype control antibodies. Figure 2D shows flow cytometry data of AIQ-HLA-A2 expression in OCI-AML3, T2, GMB, and PC-3 cells. Darkly shaded histograms are stained with YG1 scFv-Fc and anti-HA, and lightly shaded histograms are stained with BSA followed by anti-HA. Representative data from triple measurements are shown. [Figure 2-3] Figures 2A–2E show the specific and high-affinity binding of YG1 scFv to the AIQ-HLA-A2 complex on AML cells. Figure 2E shows the kinetic analysis of the interaction between YG1 scFv-Fc and AIQ-HLA-A2, SLL-HLA-A2, or HLA-A2 using biolayer interferometry. A streptavidin biosensor chip from ForteBio Octet RED 96 was coated with biotinylated YG1 scFv-Fc protein. The chip was immersed in increasing concentrations of AIQ-HLA-A2, SLL-HLA-A2, or HLA-A2 (shown below the binding curve) to measure binding (association) to scFv-Fc, and then transferred to a buffer-containing well to measure the dissociation rate (dissociation). Representative data from three separate experiments are shown. [Figure 3-1] Figures 3A-3D illustrate the generation of AIQ-HLA-A2 complex-specific NPM1c-CAR-T cells (including YG1 scFv). Figure 3A shows a schematic of a CAR vector consisting of scFv (YG1 or CD19), CD8α extracellular hinge and transmembrane domain, 4-1BB costimulatory domain, and CD3ζ activation domain, followed by self-cleaving P2A and EGFP. Figure 3B shows a schematic of the recognition of the AIQ-HLA-A2 complex on AML cells by NPM1c CAR-T cells. [Figure 3-2]Figures 3A-3D show the generation of AIQ-HLA-A2 complex-specific NPM1c-CAR-T cells (including YG1 scFv). Figure 3C shows flow cytometry data of CAR expression by non-transduced and transduced T cells. Transduced T cells were enriched and enlarged by GFP+ cell sorting and stained with AF647-labeled anti-human IgG heavy and light chain antibodies that recognize scFv. Non-transduced T cells were activated and enlarged without sorting. Staining profiles of viable cells (DAPI-) with GFP and anti-human IgG are shown. Figure 3D shows flow cytometry data demonstrating that NPM1c CAR-T cells recognize the AIQ-HLA-A2 complex. Non-transduced and transduced T cells were incubated with biotinylated AIQ-HLA-A2, SLL-HLA-A2, or HLA-A2 complexes and subsequently stained with streptavidin-APC. The staining profiles of viable (DAPI-) non-transduced T cells, NPM1c CAR-T cells, and CD19 CAR-T cells with GFP and streptavidin-APC are shown. The data in Figures 3C and 3D are representative of at least three independent experiments. Percentages indicate the percentage of cells in the gated region. [Figure 4-1]Figures 4A-4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4A-4B show that NPM1c CAR-T cells kill target cells in vitro. NPM1c CAR-T cells were co-cultured with OCI-AML3, GMB, and PC-3 tumor cells for 24 hours at the indicated effector:target (E:T) ratio. The cell mixture was stained with CD8 and CD33, CD19, or mCherry, followed by flow cytometry. The percentage of CAR-T cells was quantified by CD8 staining, the percentage of OCI-AML3 cells by CD33, the percentage of GMB cells by CD19, and the percentage of PC-3 cells by mCherry. The percentage of specific lysis of tumor cells was calculated (see Materials and Methods in the Examples section for the calculation formula). Figure 4A shows the staining profiles of CD33, CD19, or mCherry for CD8 at different E:T ratios, and the percentage of specific lysis (Figure 4B). The percentage of cells in the gated region is shown. The p-values ​​indicate a comparison between NPM1c CAR-T cells and non-transduced T cells at the same E:T ratio. [Figure 4-2] This is Figure 4A (continued). [Figure 4-3] This is Figure 4A (continued). [Figure 4-4] This is Figure 4A (continued). [Figure 4-5]Figures 4A-4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4A-4B show that NPM1c CAR-T cells kill target cells in vitro. NPM1c CAR-T cells were co-cultured with OCI-AML3, GMB, and PC-3 tumor cells for 24 hours at the indicated effector:target (E:T) ratio. The cell mixture was stained with CD8 and CD33, CD19, or mCherry, followed by flow cytometry. The percentage of CAR-T cells was quantified by CD8 staining, the percentage of OCI-AML3 cells by CD33, the percentage of GMB cells by CD19, and the percentage of PC-3 cells by mCherry. The percentage of specific lysis of tumor cells was calculated (see Materials and Methods in the Examples section for the calculation formula). Figure 4A shows the staining profiles of CD33, CD19, or mCherry for CD8 at different E:T ratios, and the percentage of specific lysis (Figure 4B). The percentage of cells in the gated region is shown. The p-values ​​indicate a comparison between NPM1c CAR-T cells and non-transduced T cells at the same E:T ratio. [Figure 4-6] Figures 4A–4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4C shows a comparison of IFN-γ and IL-2 expression between CAR-T cells and untransduced T cells. NPM1c CAR-T cells and untransduced T cells were co-cultured with OCI-AML3, GMB, or PC-3 for 12 hours in the presence of monensin and brefeldin A. Cells were stained for CD3, then permeabilized, stained for intracellular IFN-γ or IL-2, and subsequently flow cytometry was performed. Percentages of IFN-γ+ or IL-2+ NPM1c CAR-T cells and untransduced T cells are shown. P values ​​are also shown. [Figure 4-7]Figures 4A–4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4D shows that NPM1c CAR-T cells secrete various cytokines when stimulated by NPM1c+HLA-A2+ target cells. NPM1c CAR-T cells or non-transduced T cells were co-cultured with NPM1c+HLA-A2+OCI-AML3 cells for 16 hours. Culture supernatants were collected and simultaneously assayed for 20 different cytokines using the Quanibody Human Cytokine Array. Each cytokine contained four antibody spots for quadruple assay. T cells from four different healthy donors were analyzed individually. n=4 repeated antibody spots. Bars and error bars in the graphs represent mean ± se. p-values ​​are shown. [Figure 4-8] This is Figure 4D (continued). [Figure 4-9] Figures 4A–4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4E–4F show that NPM1c CAR-T cells proliferate in response to NPM1c+HLA-A2+ target cells. NPM1c CAR-T cells or non-transduced T cells were co-cultured with OCI-AML3 cells for 5 days. The absolute number of CAR-T cells or non-transduced T cells was determined by flow cytometry using precision count beads. Ki-67 expression by NPM1c CAR-T cells or non-transduced T cells was assayed by intracellular staining followed by flow cytometry. Figure 4E provides a comparison of the number of NPM1c CAR-T cells and non-transduced T cells on day 5. Figure 4F provides a comparison of the mean fluorescence intensity (MFI) of intracellular Ki-67 staining between untransduced T cells and NPM1c CAR-T cells. The p-value indicates the comparison between NPM1c CAR-T cells and untransduced T cells. The sample size was 5 biologically independent samples (n=5). The bars and error bars in the graph represent mean ± se. [Figure 4-10]Figures 4A-4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figures 4G-4H show that NPM1c CAR-T cells specifically kill HLA-A2+NPM1c+ human tumor cells in vitro. OCI-AML2 (HLA-A2 positive) and PC-3 (HLA-A2 negative) cells were transduced with lentivirus expressing NPM1c (lenti-NPM1c) or an empty negative control (lenti-NC). Transduced cells were sorted and augmented. A comparison of specific killing of transduced or control OCI-AML2 cells (Figure 4G) and PC-3 cells (Figure 4H) by NPM1c CAR-T cells or non-transduced T cells is shown. NPM1c CAR-T cells or non-transduced T cells were co-cultured for 24 hours with transduced or control OCI-AML2 or PC-3 target cells at the displayed E:T ratio. Target cell elimination was measured by assaying the luciferase activity of viable target cells. The percentage of specific lysis of tumor cells at different E:T ratios was calculated. Target cells and T cells in each response are shown. [Figure 4-11] Figures 4A-4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4I shows flow cytometry analysis of YG1 scFv-FCs bound to T2 cells pulsed with different concentrations of AIQ peptide (left panel) or SLL peptide (right panel). [Figure 4-12]Figures 4A–4J show that NPM1c CAR-T cells (including YG1 scFv) specifically kill HLA-A2+NPM1c+ human AML cells in vitro. Figure 4J shows a comparison of specific killing by NPM1c CAR-T cells or non-transduced T cells of T2 cells pulsed with different concentrations of AIQ peptide (left panel) or SLL peptide (right panel). NPM1c CAR-T cells or non-transduced T cells were co-cultured for 24 hours with T2 target cells pulsed with the peptide at the E:T ratio shown. Killing of target cells was measured by assaying the luciferase activity of viable target cells. The p-value shows the comparison between NPM1c CAR-T cells and non-transduced T cells at the same E:T ratio. n=3 biologically independent samples. Data points and error bars represent mean ± se. [Figure 5-1] Figures 5A-5H show that NPM1c CAR-T (including YG1 scFv) therapy reduces leukemia burden and extends survival in mice with NPM1c-positive AML cells, but not in mice with NPM1c-negative AML cells. Figure 5A shows the experimental design. NSG mice were intravenously injected with OCI-AML3 cells (1 × 10⁶) or GMB cells (2 × 10⁶) (D-4), and engraftment was imaged 4 days later (D0). Subsequently, the mice were intravenously injected with 1 × 10⁷ NPM1c CAR-T cells, untransduced T cells, or CD19 CAR-T cells. Mice were monitored every 3 days by bioluminescence imaging to assess tumor burden and survival. [Figure 5-2]Figures 5A-5H show that NPM1c CAR-T (including YG1 scFv) therapy reduces leukemia burden and extends survival in mice with NPM1c-positive AML cells, but not in mice with NPM1c-negative AML cells. Figure 5B shows a comparison of OCI-AML3 leukemia burden, measured by bioluminescence imaging, at the indicated days (D0-D18) after T cell injection between mice treated with NPM1c CAR-T cells and mice treated with non-transduced T cells (n=5). A comparison of total flux (luciferase signaling from systemic OCI-AML3 leukemia cells) for each experimental group is shown. The experiment was repeated twice with 4 or 5 mice per group. Figure 5C shows Kaplan-Meier survival curves (n=9) for mice treated with NPM1c CAR-T cells or non-transduced T cells as in Figure 5B. P-values ​​are shown. [Figure 5-3]Figures 5A-5H show that NPM1c CAR-T (including YG1 scFv) therapy reduces leukemia burden and extends survival in mice with NPM1c-positive AML cells, but not in mice with NPM1c-negative AML cells. Figure 5D shows a comparison of GMB lymphoma burden, measured by bioluminescence imaging on the indicated days (D0-D21) after T cell injection, between mice treated with NPM1c CAR-T cells and non-transduced T cells and mice treated with CD19 CAR-T cells (n=3-5). A comparison of total flux (luciferase signaling from whole-body GMB cells) for each experimental group is shown. The experiment was repeated twice with 3-5 mice per group. The p-values ​​for the total flux were p=0.992 for untransduced T cells and NPM1c CAR-T cells, p=0.003 for CD19 CAR-T cells and untransduced T cells, and p=0.047 for CD19 CAR-T cells and NPM1c CAR-T cells. Figure 5E shows the Kaplan-Meier survival curves (n=3-5) for mice treated with NPM1c CAR-T cells, untransduced T cells, or CD19 CAR-T cells, as shown in Figure 5D. P-values: p=0.124 for untransduced T cells and NPM1c CAR-T cells, p=0.012 for CD19 CAR-T cells and untransduced T cells, and p=0.015 for CD19 CAR-T cells and NPM1c CAR-T cells. [Figure 5-4]Figures 5A–5H show that NPM1c CAR-T (including YG1 scFv) therapy reduces leukemia burden and extends survival in mice with NPM1c-positive AML cells, but not in mice with NPM1c-negative AML cells. Figure 5F provides a comparison of OCI-AML3 leukemia burden measured by bioluminescence imaging at indicated days (D0–D21) after T cell / PBS injection in mice administered NPM1c CAR-T cells, untransduced T cells, or PBS (n=3–4). A comparison of total flux (luciferase signaling from systemic OCI-AML3 cells) is shown. The p-values ​​are PBS vs. untransduced T p=0.395, PBS vs. NPM1c CAR-T p=0.018, and untransduced T vs. NPM1c CAR-T p=0.011. Figure 5G provides a comparison of OCI-AML2 leukemia burden, measured by bioluminescence imaging on the indicated days (D0-D21) after T cell injection, between mice treated with NPM1c CAR-T cells (n=5) and mice treated with untransduced T cells (n=5). A comparison of total flux (luciferase signaling from systemic OCI-AML2 cells) is shown. Figure 5H provides Kaplan-Meier survival curves for the mice shown in Figure 5G, treated with either NPM1c CAR-T cells (n=5) or untransduced T cells (n=5). Data points and error bars represent mean ± se. p-values ​​are shown. [Figure 6-1] Figures 6A–6I show that NPM1c CAR-T cells (including YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figure 6A shows a comparison of OCI-AML3 leukemia burden as measured by bioluminescence imaging between NSG mice injected with OCI-AML3 AML cells and then untransduced T cells, or NSG mice injected with OCI-AML3 AML cells and then NPM1c CAR-T cells. A comparison of total flux (luciferase signaling from OCI-AML3 cells throughout the body) is shown. Mice (n=5) were imaged on the day of T cell injection (day 0) and 18 days later. [Figure 6-2]Figures 6A-6I show that NPM1c CAR-T cells (including YG1 scFv) reduce leukemia burden in blood, spleen, bone marrow, and liver. Figures 6B-6C show representative flow cytometry plots of the gating strategy and expression profile for the cell population obtained from the mice represented in Figure 6A. Blood, spleen, bone marrow, and liver were collected on day 18 to prepare single cell suspensions, which were stained for mouse CD45 as well as human CD45, CD8, CD33, PD-1, and Tim-3, followed by flow cytometry. Representative staining profiles and gating strategies for blood and spleen (Figure 6B) and bone marrow and liver (Figure 6C) are shown. The gating strategy includes gating of mCD45 vs. hCD45 in live cells (DAPI-), gating of hCD33 vs. hCD8 in hCD45+ cells, gating of hPD-1 vs. hCD8 in hCD8+ cells, and gating of hTim-3 vs. hCD8 in hCD8+ cells. The numbers indicate the percentage of cells in the gated regions. [Figure 6-3] Figures 6A-6I show that NPM1c CAR-T cells (including YG1 scFv) reduce leukemia burden in blood, spleen, bone marrow, and liver. Figures 6B-6C show representative flow cytometry plots of the gating strategy and expression profile for the cell population obtained from the mice represented in Figure 6A. Blood, spleen, bone marrow, and liver were collected on day 18 to prepare single cell suspensions, which were stained for mouse CD45 as well as human CD45, CD8, CD33, PD-1, and Tim-3, followed by flow cytometry. Representative staining profiles and gating strategies for blood and spleen (Figure 6B) and bone marrow and liver (Figure 6C) are shown. The gating strategy includes gating of mCD45 vs. hCD45 in live cells (DAPI-), gating of hCD33 vs. hCD8 in hCD45+ cells, gating of hPD-1 vs. hCD8 in hCD8+ cells, and gating of hTim-3 vs. hCD8 in hCD8+ cells. The numbers indicate the percentage of cells in the gated regions. [Figure 6-4] Figures 6A-6I show that NPM1c CAR-T cells (including YG1 scFv) reduce leukemia burden in blood, spleen, bone marrow, and liver. Figure 6D shows a comparison of the total numbers of hCD33+ leukemia cells and hCD8+ T cells in different tissues between mice administered NPM1c CAR-T cells and non-transduced T cells (the left bar represents treatment with non-transduced T cells, and the right bar represents treatment with NPM1c CAR-T cells). [Figure 6-5] Figures 6A-6I show that NPM1c CAR-T cells (including YG1 scFv) reduce leukemia burden in blood, spleen, bone marrow, and liver. Figure 6E shows a comparison of the percentages of hCD33+ leukemia cells and hCD8+ T cells among hCD45+ cells in different tissues between mice administered NPM1c CAR-T cells and mice administered non-transduced T cells (the left bar represents treatment with non-transduced T cells, and the right bar represents treatment with NPM1c CAR-T cells). [Figure 6-6] Figures 6A-6I show that NPM1c CAR-T cells (including YG1 scFv) reduce leukemia burden in blood, spleen, bone marrow, and liver. Figure 6F shows the ratio of the percentage of hCD8+ T cells to hCD33+ leukemia cells in different tissues of mice administered NPM1c CAR-T cells or non-transduced T cells (the left bar represents treatment with non-transduced T cells, and the right bar represents treatment with NPM1c CAR-T cells). Figures 6G-6H show the percentages of PD1+ T cells (Figure 6G) or Tim-3+ T cells (Figure 6H) among human CD8+ T cells in different tissues (the left bar represents treatment with non-transduced T cells, and the right bar represents treatment with NPM1c CAR-T cells). P values are shown in Figures 6A, 6D, 6E, 6F, 6G, and 6H (n = 5). [Figure 6-7]Figures 6A–6I show that NPM1c CAR-T cells (including YG1 scFv) reduce leukemia burden in the blood, spleen, bone marrow, and liver. Figure 6I shows that NPM1c CAR-T cells effectively eliminate leukemia cells in the bone marrow 30 days after CAR-T cell injection. NSG mice were transplanted with OCI-AML3 and injected with NPM1c CAR-T cells, untransduced T cells, or PBS 4 days later (mice are shown in Figure 5F). Cells were collected from the bone marrow of viable mice 30 days after T cell injection. Cells were stained for mouse CD45 and human CD45, CD8, and CD33, followed by flow cytometry. Staining profiles for FSC and DAPI of whole cells (left), staining profiles for gated hCD45 and mCD45 of viable (DAPI-) cells (middle), and staining profiles for hCD33 and hCD8 of hCD45+ cells (right) are shown. The numbers indicate the percentage of cells within the gated region. [Figure 6-8] Figure 6I (continued). [Figure 6-9] Figure 6I (continued). [Figure 6-10] Figure 6I (continued). [Figure 7-1] Figures 7A–7G show that NPM1c CAR-T cells (including YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo, but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7A shows that NPM1c CAR-T cells kill NPM1c+HLA-A2+ primary AML blast cells from three donors in vitro. NPM1c CAR-T cells or non-transduced T cells were incubated with AML blast cells in the ratios shown for 24 hours. The absolute number of AML blast cells was quantified by staining for CD8 and CD33, followed by flow cytometry using precision count beads. The percentage of specific lysis of tumor cells at different E:T ratios was calculated. Biological replicates of n=3. Bars and error bars in the graphs represent mean ± se. p-values ​​are shown. [Figure 7-2] Figures 7A–7G show that NMP1c CAR-T cells (including YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo, but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7B shows flow cytometry analysis of HLA-A2 expression by HSPCs. Human CD34+ HSPCs were purified from the livers of two donor fetuses using the EasySep Human CD34 Positive Selection Kit. Dark histograms: stained with anti-HLA-A2, and light histograms: stained with isotype control antibody. Representative data from technical triplicates are shown. Figure 7C shows flow cytometry analysis of HSPCs for YG1 scFv-Fc binding. Dark histograms: stained with YG1 scFv-Fc and anti-HA, light histograms: stained with BSA and then anti-HA. Representative data from three individual experiments using technical triplicate measurements are shown. [Figure 7-3] Figures 7A–7G show that NPM1c CAR-T cells (including YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo, but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7D shows that NPM1c CAR-T cells do not kill HLA-A2+CD34+ HSPCs. NPM1c CAR-T cells and untransduced T cells were incubated for 24 hours at an E:T ratio labeled as HSPC. The cell mixture was stained for CD8 and CD34 and quantified by flow cytometry using precision count beads. Examples of staining profiles for CD8 (T cells) and CD34 (HSPCs) at different E:T ratios are shown. Percentage of cells within the gated region is shown. [Figure 7-4]Figures 7A–7G show that NMP1c CAR-T cells (including YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo, but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7E shows a comparison of specific cytolysis between NMP1c CAR-T cells and untransduced T cells at different E:T ratios. The p-values ​​indicate the comparison between NMP1c CAR-T cells and untransduced T cells at the same E:T ratio. Biological replicates of n=3. Data points and error bars represent mean ± se. [Figure 7-5] Figures 7A–7G show that NPM1c CAR-T cells (including YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo, but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7F shows that treatment with NPM1c CAR-T cells reduces the leukemia burden in primary HLA-A2+NPM1c+AML xenograft. Human AML blast cells were transplanted into NSGS mice. After 2 weeks, when AML blast cells were detectable in the blood, the mice were administered NPM1c CAR-T cells or non-transduced T cells. Blood was collected from the mice on the indicated day after T cell transfer, and mononuclear cells were stained for mCD45, hCD45, and hCD8. Representative staining profiles of hCD45 and mCD45, gated for hCD8- viable cells, are shown. AML blast cells were hCD45+hCD8-. The numbers indicate the percentage of cells within the gated region. [Figure 7-6]Figures 7A–7G show that NPM1c CAR-T cells (including YG1 scFv) effectively kill primary human AML blast cells in vitro and in vivo, but do not exhibit cytotoxicity against normal human HLA-A2+CD34+ hematopoietic stem / progenitor cells (HSPCs). Figure 7G shows a comparison of the percentage of hCD45+CD8-AML blast cells in peripheral blood between mice treated with NPM1c CAR-T cells and mice treated with untransduced T cells. AML blast cell levels were measured before T cell injection (day 0) and at days 9 and 18 after T cell injection. The left bar represents treatment with untransduced T cells, and the right bar represents treatment with NPM1c CAR-T cells. The bars and error bars in the graph represent mean ± se. The p-value (two-sided independent sample t-test) is shown (n=5). [Modes for carrying out the invention]

[0063] This disclosure relates, at least in part, to NPM1c neoepiform complexes with HLA-A2. To identify single-chain variable antibody fragments (scFv) that bind specifically and with high affinity to topes. Based on this disclosure, such NPM1c neoepitotes that form complexes with HLA-A2 This disclosure provides a novel scFv that binds to a p, an antibody, and its antigen-binding fragment. In addition, this disclosure provides , specifically binds to such NPM1c neoepitopes that form complexes with HLA-A2. This disclosure provides a bispecific binding molecule based on scFv that further binds to other targets. do.

[0064] Furthermore, this disclosure includes NPM1c neoepitopes that form complexes with HLA-A2. Chimeric antigen receptor (CAR) polypeptides containing extracellular binding domains that specifically bind to antigens We will provide Chido.

[0065] Furthermore, this disclosure includes NPM1c neoepitopes that form complexes with HLA-A2. T cells expressing CAR polypeptides containing an extracellular binding domain that specifically binds to an antigen. Provides, as described herein and shown in the examples presented herein, It specifically binds to antigens containing the NPM1c neoepitope, which forms a complex with HLA-A2. T cells expressing CAR polypeptides containing an extracellular binding domain are in vitro This specifically kills AML cells and, in vivo, leukemia is suppressed in an AML mouse model. It reduces the amount and extends the survival period.

[0066] Therefore, antibodies and their antigen-binding fragments, bispecific molecules, CAR polypeptides, Furthermore, T cells expressing the CAR polypeptides described herein are affected by the NPM1c mutation. It is useful for targeted immunotherapy to treat existing cancers. For example, in this specification Disclosed are an antibody and its antigen-binding fragment, a CAR polypeptide, and a CAR polypeptide The expressed T cells are used for targeted immunotherapy to treat acute myeloid leukemia (AML). It is useful for the following. In one embodiment, an antigen containing an NPM1c neoepitope is used. The 'slope' is a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A). 2) An antibody that specifically binds when it forms a complex with (or is presented by) the above. Or the antigen-binding fragment thereof is provided herein. In one embodiment, the following amino acids Sequence: AIQDLCLAV (sequence number 1), AIQDLCVAV (sequence number 71), CL AVEEVSL (sequence number 72), VEEVSLRK (sequence number 73), AVEEVSL R (sequence number 74), AVEEVSLRK (sequence number 75), CLAVEEVSLRK ( One or more of the neoepitopes having SEQ ID NO: 76), such an epitope Specifically binds when the antibody or its antigen-binding fragment forms a complex with a class I major histocompatibility complex (MHC class I) protein (e.g., HLA-A2 ), is provided herein. In one aspect, an antibody or its antigen-binding fragment that does not bind or substantially bind to the MHC class I protein alone is provided herein . In one aspect, an antibody or its antigen-binding fragment that does not bind or substantially bind to a control peptide (e.g., the control peptide is a NY-ESO-1 epitope (e.g., a peptide containing SEQ ID NO: 62) or an influenza virus M1 epitope (e.g., a peptide containing SEQ ID NO: 63)) that forms a complex with the MHC class I protein is provided herein . In one aspect, an antibody or its antigen-binding fragment that does not bind or substantially bind to the NPM1c neoepitope alone (without the MHC class I protein) is provided herein. In some aspects, the NPM1c neoepitope contains the amino acid sequence AIQDLCLAV (SEQ ID NO: 1), and the MHC class I protein is the HLA-A2 protein (e.g., the protein encoded by the HLA-A*02:01 allele). In some aspects, the antigen is on the surface of cancer cells (e.g., when the cancer is NPM1c+, e.g., when the cancer is AML). In one aspect, the HL A-A2 protein (e.g., the protein encoded by the HLA-A*02:01 allele) specifically binds to the amino acid sequence containing AIQDLCLAV (SEQ ID NO: 1) that forms a complex with it . In some aspects, the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, a single-chain Fv fragment, a diabody, a triabody, a minibody, a nanobody, or a complementarity-determining region (CDR) grafted antibody. In some aspects, the antibody is a monoclonal antibody, a polyclonal antibody, or a chimeric antibody. In some aspects, the antibody is conjugated to a detectable label, a therapeutic agent, or a cytotoxic agent. In some aspects, the antibody is produced by a recombinant DNA technology, a phage display technology, or a transgenic animal technology. In some aspects, the antigen is present in a cancer cell line, a cancer tissue sample, or a patient sample. In some aspects, the cancer is NPM1c+, e.g., when the cancer is AML. In one aspect, the HLA-A2 protein (e.g., the protein encoded by the HLA-A*02:01 allele) forms a complex with the AIQDLCLAV (SEQ ID NO: 1) containing amino acid sequence specifically binds to the amino acid sequence containing AIQDLCLAV (SEQ ID NO: 1) that forms a complex with it Antibodies or antigen-binding fragments thereof that bind heteroto are provided herein. The antibodies and their antigen-binding fragments provided are described below.

[0067] In one embodiment, (i) Class I major histocompatibility complex (MHC class I) proteins (for example) Furthermore, it specifically targets antigens containing the NPM1c neoepitope, which forms a complex with HLA-A2. (ii) a first binding domain that binds to the second antigen, and (ii) a second antigen that specifically binds to the second antigen. Bispecific molecules containing a binding domain are provided herein. In some embodiments, The second antigen is an antigen expressed on T cells or natural killer cells. In this case, the second antigen is CD3 (e.g., human CD3), NKp46 (e.g., human NK p46), or CD16A (e.g., human CD16A). In one embodiment, (i)H LA-A2 protein (for example, the protein encoded by the HLA-A*02:01 allele) The amino acid sequence containing AIQDLCLAV (SEQ ID NO: 1) that forms a complex with protein. (ii) a first antigen-binding domain that specifically binds to the antigen, and (ii) a second antigen that specifically binds to the antigen. A bispecific molecule comprising a second antigen-binding domain is provided herein. The bispecific molecules provided in the specification are described below.

[0068] In one embodiment, the antibody or its antigen-binding fragment described herein (and optionally, A pharmaceutical composition comprising a pharmaceutically acceptable carrier is provided herein. In one embodiment This refers to the bispecific molecules described herein (and optionally, pharmaceutically acceptable carriers). A pharmaceutical composition comprising ) is provided herein. Details are provided below.

[0069] In one embodiment, a chimeric antigen comprising an intracellular domain, a transmembrane domain, and an extracellular domain. A receptor (CAR) polypeptide in which the extracellular domain is a class I major histocompatibility complex NPM1 that forms a complex with a body (MHC class I) protein (e.g., HLA-A2) A CAR polypeptide that specifically binds to an antigen containing a neoepitope is specified herein. It is provided as follows. In one embodiment, it includes an intracellular domain, a transmembrane domain and an extracellular domain. A CAR polypeptide in which the extracellular domain is an HLA-A2 protein (for example, It formed a complex with the protein encoded by the HLA-A*02:01 allele. AIQDLCLAV (SEQ ID NO: 1) is a CAR poly(Among amino acid sequences) that specifically binds to amino acid sequences. Peptides are provided herein. In one embodiment, an intracellular domain, a transmembrane domain and a CAR polypeptide comprising an extracellular domain, wherein the extracellular domain is specified A CAR polypeptide comprising any of the antibodies or antigen-binding fragments described herein is Provided in detail. In one embodiment, intracellular domain, transmembrane domain and extracellular domain A CAR polypeptide comprising a main, wherein the extracellular domain is as described herein. A CAR polypeptide comprising either of the bispecific molecules is provided herein. The CAR polypeptides provided in the specification are described below.

[0070] In one embodiment, a CAR polymorphic includes an intracellular domain, a transmembrane domain, and an extracellular domain. Immunoeffector cells expressing peptides, wherein the extracellular domain is a class I major group It forms complexes with MHC class I proteins (e.g., HLA-A2). Immune effector cells that specifically bind to antigens containing the NPM1c neoepitope are Provided in the specification. In one embodiment, intracellular domain, transmembrane domain and extracellular domain. Immuneffector cells expressing CAR polypeptides containing a domain, and extracellular domain The main component is the HLA-A2 protein (for example, depending on the HLA-A*02:01 allele). AIQDLCLAV (SEQ ID NO: 1), which forms a complex with the protein being included. Immunoeffector cells that specifically bind to an amino acid sequence are provided herein. In one embodiment, a CAR polymorphic molecule containing an intracellular domain, a transmembrane domain, and an extracellular domain is used. An immunoeffector cell expressing a peptide, wherein the extracellular domain is as described herein. Immunoeffector cells containing any of the antibodies or antigen-binding fragments thereof are specified herein. It is provided in one embodiment, an intracellular domain, a transmembrane domain and an extracellular domain. An immune effector cell expressing a CAR polypeptide containing the extracellular domain However, immune effector cells containing any of the bispecific molecules described herein are Provided in the specification. In one embodiment, the CAR polypeptide described herein is generated Immunoeffector cells that exhibit this property are provided herein. In one embodiment, immunoeffector T cells are T cells (e.g., CD8 + T cells, natural killer cells, or macromolecules It is a lophage. In one embodiment, the expression of CAR polypeptides affects immune effector cells. NPM that forms complexes with Class I major histocompatibility complex (MHC class I) proteins Cancer cells that display a 1c neoepitope (e.g., HLA-A2) on their surface (e.g., The cancer is targeted (AML). In one embodiment, the expression of CAR polypeptides is immune Effector cells are affected by HLA-A2 protein (e.g., HLA-A*02:01 allele). The amino acid sequence AIQDLCLAV forms a complex with the protein encoded by Targeting cancer cells that display (SEQ ID NO: 1) on their surface (e.g., cancer is AML). To cause. The immune effector cells provided herein are described below.

[0071] In one embodiment, any of the immunoeffector cells described herein (and optional selections) Therefore, pharmaceutical compositions comprising a pharmaceutically acceptable carrier are provided herein.

[0072] In one embodiment, a method for treating cancer in a subject (e.g., a human), including cancer The cell surface of a cell contains Class I major histocompatibility complex (MHC class I) proteins (for example) This paper presents an NPM1c neoepitope that forms a complex with HLA-A2, and describes it herein. A method comprising the step of administering any of the listed antibodies or antigen-binding fragments is described in this Provided in the specification. In one embodiment, the treatment of cancer in a subject (e.g., a human) A method in which the cell surface of cells including cancer cells is determined to be a Class I major histocompatibility complex (MHC class I) NPM1c neoepitol that forms a complex with a protein (e.g., HLA-A2) The presenter presents any immunoeffector containing the CAR polypeptide described herein. A method comprising the step of administering a target cell is provided herein. In one embodiment This is a method for treating cancer in a subject (for example, a human), and the cells of the cancer include cells. The surface is coated by the HLA-A2 protein (for example, the HLA-A*02:01 allele). AIQDLCLAV (SEQ ID NO: 1), which forms a complex with the protein being treated, contains The amino acid sequence is presented and targets any of the antibodies or antigen-binding fragments described herein. A method comprising the step of administering is provided herein. In one embodiment, the subject (for example) For example, a method for treating cancer in humans, wherein the cell surface of cells containing cancer is HLA -A2 protein (for example, the protein encoded by the HLA-A*02:01 allele) The amino acid sequence containing AIQDLCLAV (SEQ ID NO: 1), which forms a complex with the protein, is presented. This indicates any immune effector cell containing the CAR polypeptide described herein. A method comprising the step of administering to a target is provided herein.

[0073] In some embodiments, methods for treating NPM1c-positive cancer in subjects (e.g., humans) There is a steroid that is administered targeting any of the antibodies or antigen-binding fragments described herein. Methods including a sprocket are provided herein. In some embodiments, the subject (e.g., human) A method for treating NPM1c-positive cancer in ) the CARP described herein A method comprising the step of administering any immunoeffector cells containing a lipeptide to a target. However, this is provided herein.

[0074] In one embodiment, a method for treating AML in a subject (e.g., a human), as specified herein A method comprising the step of administering any of the antibodies or antigen-binding fragments described herein This is provided herein. In one embodiment, AML is treated in a subject (e.g., a human). A method of treatment comprising any immunoassay comprising a CAR polypeptide as described herein. A method comprising the step of administering target cells is provided herein.

[0075] Antibodies or antigen-binding fragments provided herein, CAR polysynthetic compounds provided herein Peptides, immune effector cells containing CAR polypeptides provided herein, and Methods of treatment using pharmaceutical compositions containing the same provided herein, and the use thereof. Kits that include these items are listed below.

[0076] This disclosure also relates, at least in part, to MHC proteins (in particular, MHC class I proteins) Neoepitopes (especially cancer neoepidermal cells) that form complexes with proteins such as HLA-A2. This is based on the identification of scFv that specifically bind to tope. The isolation of the antibody indicates that it is an antibody that specifically binds to the neoepitope-MHC complex and This was surprising, given the many challenges involved in developing the antigen-binding fragment. In particular, Oepitope peptides readily dissociate from MHC proteins, and this is what causes them to form this complex. This makes it difficult to produce specific antibodies. It is not bound by any theory, but yeast Using the mother surface display method, MHC proteins alone or in complex with a control peptide were displayed. The neoepitope-MHC complex is specific to the formed MHC protein without binding to it. By using multiple positive and negative selections of yeast populations that bind to neoepitope- This discovery led to the identification of scFv that specifically bind to MHC complexes. The steps are described in Example 1 and Annex 1. Not bound by any theory. However, dimeric neoepitope-MHC complexes have also been used as antigens, and high affinity... This may have contributed to the successful isolation of specific scFv.

[0077] Therefore, in one embodiment, MHC (e.g., MHC class I) proteins (e.g., H neoepitopes that form complexes with (or are presented by) LA-A2 (e.g.) For example, an antibody or its antigen-binding fragment that specifically binds to an antigen (including a cancer neoepitope). However, this is provided herein. In certain embodiments of this model, an antibody or antigen The binding fragment can be used to display a yeast surface display antibody (e.g., scFv) library or fur. Generated using a didisplay antibody (e.g., scFv) library, and optionally Multiple (2, 3, 4 or more) positive selections (yeast that binds to the antigen) (or select a population of phages) and multiple times (2, 3, 4 or more times) ) Negative selection (e.g., MHC protein alone and / or control peptide (i.e., yeast that binds to MHC proteins that have formed a complex with a peptide (different from the neoepitope) Neoepitope-MHC complexes (by selecting and removing a population of mother or phages) The specific binder for the body is concentrated. In a particular embodiment of this aspect, the antibody and The antigen-binding fragment is a yeast surface display antibody (e.g., scFv) library or Generated using a phage display antibody (e.g., scFv) library, optional In the selection process, multiple positive selections (at least 3 times, at least 4 times, or at least 5 times) Select a population of yeast or phages that bind to the antigen) and multiple times (at least twice, Negative selection (e.g., MHC protein alone) at least 3 times, or at least 4 times Yeast or f Selectively remove a group of phages (for example, yeast that binds to MHC proteins alone or Selectively remove the phage population at least twice and form a complex with the control peptide. Select a population of yeast or phage that binds to the resulting MHC protein at least twice. Using (to remove), a specific binder for the neoepitope-MHC complex is used. The cells are then concentrated. In a particular embodiment, the cell population is then (for example, 1) after the selection step. After one or more positive selection steps, after one or more negative selection steps or increased after each selection step. In certain embodiments of this model, the antigen This refers to dimeric neoepitope-MHC complexes (especially, for example, IgG Fc, for example, MHC). (Having two neoepitopes linked via human IgG1 or human IgG1: MHC molecules) In certain embodiments, yeast display methods and libraries are used. In certain embodiments, positive selection is performed on both antigens (neoepitope-MHC complex) and selection of a population of yeast or phage to be stained for the antibody (e.g., scFv) For example, including at least one or at least two selections. In a particular embodiment, A population of yeast or phages is labeled with antigens and antibodies / fragments (e.g., scFv). Molecular Magnetic Sorting (MACS) and / or Flow Cytometry Saw It is selected by ting. For example, the antigen is covalently bound to biotin. This is biotinylated (this is a secondary drug conjugated to a fluorophore). (for example, detectable by streptavidin), and / or IgG molecules ( This is detected by an IgG-specific labeled antibody) and is bound to the antibody (e.g., sc Fv) is an epitope tag (e.g., hemagglutinin or c-Myc) (this is an epitope It is bound to the tag (which is detected by a fluorescently labeled antibody against the tag). In one embodiment, the following Use at least 3, 4, 5, 6, 7, 8, or 9 of the selection steps. Then, an antibody or its antigen-binding fragment that specifically binds to the neoepitope-MHC complex is used. To generate: (i) Labeled neoepitope: Antibody-containing claw that binds to the MHC complex Positive selection of cells (e.g., selection via labeled neoepitope-MHC complexes) (ii) By doing so, (ii) clones / flesh possessing antibodies that bind to the control peptide-MHC complex Negative selection (selective removal) of cells (e.g., binding by a labeled control peptide-MHC complex) (iii) by selecting antibody-bearing clones / cells that do not undergo treatment, (iii) neoepitope -Positive selection of clones possessing antibodies that bind to MHC (e.g., antibody (e.g., scFv)) Clones / cells possessing antibodies that are double-positive stained for neoepitope-MHC complexes (iv) By selecting (iv) Labeled neoepitope: Binds to MHC complex Positive selection of antibody-bearing clones / cells (e.g., labeled neoepitope-MHC complexes) Select antibody-bearing clones / cells that are stained by the antibody, and optionally, stained with respect to the antibody. (v) by binding to the control peptide-MHC complex or the MHC complex alone. Negative selection (selective removal) of antibody-bearing clones / cells (e.g., labeled control peptide- Antibody-retaining clones that are not bound by MHC complexes or labeled MHC proteins (by selecting cells), (vi) possessing antibodies that bind to neoepitope-MHC Positive selection of loans (e.g., possessing antibodies that stain for neoepitope-MHC complexes) This can be done by selecting clones / cells, or by antibody (e.g., scFv) expression and Select antibody-containing clones / cells that are double-positively stained for the oepitope-MHC complex. (by doing so), (vii) the shadow of antibody-possessing clones / cells that bind to MHC complexes alone. Sex selection (selective removal) (for example, antibody retention that does not receive binding by labeled MHC proteins) (by selecting clones / cells), (viii) neoepitope-MHC complex Positive selection of antibody-possessing clones / cells that bind to the antibody (e.g., the antibody (e.g., scFv) and Antibody-containing clones / cells that are double-positive stained for neoepitope-MHC complexes (by selection), (ix) clones / cells possessing antibodies that bind to MHC complexes alone Negative selection (selective removal) (for example, not bound by labeled MHC protein) (By selecting antibody-bearing clones / cells). In some embodiments, the following are listed in the preceding sentence. Of the steps listed, at least 5, 6, 7, 8, or 9 are at least A specific antibody, comprising two positive selection steps and at least two negative selection steps. or are used in the fragment selection process. In some embodiments, at least six, seven 8 or 9 steps related to the neoepitope-MHC complex, at least 2 One (preferably at least three) positive selection steps, the control peptide-MHC complex At least one (preferably at least) for selectively removing the merging clones / cells Two negative selection steps, and selection of clones / cells that bind to MHC proteins alone. A negative selection step of at least one (preferably at least two) to remove Used in a selection process for specific antibodies or fragments, including. Specific implementation of this embodiment Morphologically, MHC proteins are MHC class I proteins (e.g., HLA-A2) Yes. In a particular embodiment of this aspect, the neoepitope is the NPM1c neoepitope. Yes. In one particular embodiment, the neoepitope:MHC complex is NPM1c:MHC This is LAS I (for example, NPM1c:HLA-A2). In some embodiments of this model, A neoepitope is any of the NPM1c epitopes referenced in this disclosure (e.g., It is an epitope having the amino acid sequence AIQDLCLAV (SEQ ID NO: 1). In one embodiment, the neoepitope:MHC complex is AIQDLCLAV:HLA-A 2. In some embodiments, an antibody specific to the neoepitope:MHC complex is used. Or the antigen-binding fragments are any four, five, six, or seven of the following listed in Annex 1 below. , obtained using eight or all of the selection steps. In some embodiments, the yeast table Instead of surface display or phage surface display methods and libraries, bacteria Display, eukaryotic virus display, mammalian cell display, or microscopic Antibody screening techniques are used on the yeast surface (e.g., ribosome display). Display methods and libraries are publicly known in the art (e.g., Chao et al., See Nature Protocols 1(2):755-768, 2006. Phage display methods and ly Blurry is well known in this art. Merz et al. (1995) J Neurosci Methods 62(1-2 ):213-9; Di Niro et al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. 1995) Methods Mol Biol 51:355-376.

[0078] antibody In one embodiment, it forms a complex with an MHC protein (e.g., an MHC class I protein). Neoepitopes (e.g., cancer neoepitopes) that have been (or are presented as) An antibody that binds to an antigen containing (for example, specifically binds to) this antigen, and its antigen-binding fragment, Provided in the specification. In certain embodiments, among the methods described herein By one of these methods (for example, by yeast surface display method, phage display method, etc.) Multiple selection steps are used to identify specific binders; optionally, dimers Using neoepitope-MHC complexes as antigens; for example, neoepitope-MHC The complex or dimeric neoepitope-MHC complex is used as an antigen to induce antibody production. MHC proteins (e.g., MH) obtained through immunotherapy performed on the target of the immunotherapy used. Neoepi (class C I protein) that forms a complex with (or is presented by) Antibodies that specifically bind to antigens containing tope (e.g., cancer neoepitope) and their anti- Original binding fragments are provided herein. In certain embodiments, as described herein MHC class I proteins (e.g., HLA) obtained by one of the methods. -A2, or proteins encoded by the HLA-A*02 allele group) and multiple Specific to antigens containing cancer neoepitopes that have formed agglomerates (or are presented thereby). Antibodies that bind to a specific target and antigen-binding fragments thereof are provided herein. In the embodiment, the anti-MHC protein does not bind to the MHC protein alone, or does not bind substantially to it. A body or its antigen-binding fragment is provided herein. In one embodiment, an MHC protein The control peptide that forms a complex with the cereal molecule does not bind, or does not bind substantially, anti- A body or an antigen-binding fragment thereof is provided herein. In one embodiment, a neoepitoto It does not bind to the protein alone (without accompanying MHC proteins), or it does not bind to it substantially. Antibodies or antigen-binding fragments thereof are provided herein.

[0079] Functional MHC class I molecules include α-heavy chains and β2-microglobulin chains. Peptide bonds formed by Class I molecules involve the peptide amino acid side chain and the α1 and α2 dorsal bonds of the heavy chain. Dispersed pockets within the peptide bond grooves of the MHC molecule formed by the main and This is achieved through the interaction of [components]. Typically, in the case of human leukocyte antigens (HLA), the main [component] is [component]. The combined energy is the B and C-terminal residues of the peptide and the MHC molecule, respectively. The interaction with the F-binding pocket is due to the side chains throughout the peptide, which contribute to the MHC binding ability. It can increase or decrease force (e.g., Guo, et al (1992) Nature 3) 60:364; Silver et al (1992) Nature 360:367; Gorga et al (1992) Proteins 12;87; M adden (1995) Annu Rev Immunol 13:587; Madden et al (1993) Cell 75;693; Madden et al al (1992) Cell 70:1035; Bjorkman, et al (1987) Nature 329:512; Saper et al (199 1) See J Mol Biol 219:277). For a 9-amino acid peptide, the C-terminal residue (position) 9) interacts with the F-binding pocket of the MHC molecule.

[0080] MHC molecules are highly polymorphic, with thousands of allele variants at class IA and B loci. They have been identified. Most of the polymorphisms are located in the peptide bond pocket, and as a result, MHC The offspring exhibits a wide range of peptide bond specificities. Despite this polymorphism, in this field... So, HLA class I molecules can be grouped into multiple groups based on their common peptide bond specificity. It is known that each group (supertype) can be grouped together. A "pepper type" is a peptide residue that is an important residue for MHC binding, or "anchor residue". It is defined by a peptide consensus sequence that reflects the position of the do. For example, A2-sequence Per-type HLA class I molecules (i.e., HLA-A2, or HLA-A*02) The protein encoded by the Rel group has a small aliphatic residue at position 2 of the peptide. Bases (for example, alanine, tyrosine, serine, valine, leucine, isoleucine, methionine) (e.g., glutamine), and aliphatic residues at the C-terminus (e.g., leucine, isoleucine, ba Peptide having phosphorus, methionine or small hydrophobic residues (e.g., alanine, valine) It has a common specific binding to cydops (e.g., Sidney, et al (2008) BMC Immunol). (See ogy 9:1).

[0081] In certain embodiments, MHC class I proteins, for example, HLA-A2 (for example) , NPM1c (HLA-A2) forms a complex (or is presented thereby) Contains a myeloid leukemia (AML)-associated mutant nucleophosmin protein neoepitope An antibody that binds to an antigen (for example, specifically) and its antigen-binding fragment, as described in this specification. It is provided in the book.

[0082] Genomic analysis of AML shows that AML has a lower mutational burden than most other adult cancers. Studies have shown that the average number of coding mutations per AML patient is 13 (Ley et al.). al., N Engl J Med 368: 2059 (2013); Alexandrov et al., NATURE 500: 415 (2013); See Kandoth et al., NATURE 502 333 (2013). However, somatic mutations in AML These are often found in the same gene (Ley et al., N Engl J Med 368: 2059 (2013); Pap See aemmanuil et al., N Engl J Med 374: 2209 (2016), therefore, these hots Neoantigens derived from tospot mutations are attractive targets for tumor-specific immunotherapy. (See van der Lee et al., J CLIN INVEST 129: 774 (2019)). The most common variants One difference is that it codes for nucleophosmin (NPM1; coded by NPM1). There is a 4-nucleotide duplication in a key driver gene, which is present in adult AML patients. Present in 30-35% of the total (Ley et al., N Engl J Med 368: 2059 (2013); Papaemm anuil et al., N Engl J Med 374: 2209 (2016); Falini et al., N Engl J Med 352: 25 See 4 (2005). Such mutations in NPM1 result in their abnormal cytoplasmic localization. This mutant protein is called NPM1c. NPM1c mutants associated with AML... The protein is HLA class I restrictive and is HLA-A*02:01 allele and other It generates leukemic neoantigens that are presented on leukemic blast cells of patients with several alleles. For example, NPM1c is the most common HLA-A*0201 allele (about 5% of the human population). 0%) presents a leukemia-specific neoantigen epitope (AIQDLCLAV(sequence) (Number 1), abbreviated as AIQ, causes (see Greiner et al., BLOOD 120: 1282 (2012)). .

[0083] In some embodiments, it forms a complex with MHC class I proteins, such as HLA-A2. Antibodies that bind to antigens including (or present by) NPM1c neoepitopes A body and its antigen-binding fragment are provided herein. NPM1c neoepitope The length is any length that is reasonable for a peptide that binds to an MHC class I molecule. In some embodiments, the length of the NPM1c neoepitope is 5-20 amino acids, 6-19 amino acids. Mino acids, 7-18 amino acids, 8-17 amino acids, 8-16 amino acids, 8-15 amino acids, 8-15 amino acids, 8-14 amino acids, 8-13 amino acids, 8-12 amino acids, 9-12 It consists of amino acids, or 9 to 11 amino acids. In some embodiments, it is an NPM1c neoepitope. The lengths are 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9. It consists of 8, 7, 6, or 5 amino acids. In some embodiments, the length of the NPM1c neoepitope. It is 12 amino acids. In some embodiments, the length of the NPM1c neoepitope is 11 It is an amino acid. In some embodiments, the length of the NPM1c neoepitope is 10 amino acids. In some embodiments, the length of the NPM1c neoepitope is 9 amino acids. Therefore, the length of the NPM1c neoepitope is 8 amino acids. In some embodiments, NP M1c neoepitopes consist of 10, 15, 20, 30, 40, 50, or 100 amino acids. Inside the polypeptide of residue length, 20, 19, 18, 17, 16, 15, 14, 13, 1 It is a peptide consisting of 2, 11, 10, 9, 8, 7, 6, or 5 consecutive amino acids.

[0084] In some embodiments, the NPM1c neoepitope is an MHC class I HLA-A2. It binds to protein. In some embodiments, NPM1c neoepitol binds to HLA-A2. P is a small aliphatic residue at position 2 of the amino acid sequence (e.g., alanine, tyrosine, serine). (valine, leucine, isoleucine, methionine, glutamine) and amino acid sequence The C-terminal residue is an aliphatic residue (e.g., leucine, isoleucine, valine, methionine) It contains amino acid sequences, which are small hydrophobic residues (e.g., alanine, valine). In this embodiment, the NPM1c neoepitope that binds to HLA-A2 is located at the position of the amino acid sequence. 2 is valine, isoleucine, or leucine, and the C-terminal residue of the amino acid sequence is valine It contains an amino acid sequence that is leucine or isoleucine. NPM1c NeoEpitone In some embodiments, where the length of the 'p' is 8 amino acid residues, the C-terminal amino acid is at position 8. NPM In some embodiments, where the 1c neoepitope is 9 amino acid residues long, the C-terminal amino acid is located 9. In some embodiments, the NPM1c neoepitope is 10 amino acid residues long, C The terminal amino acid is at position 10. The NPM1c neoepitope is 11 amino acid residues long. In some embodiments, the C-terminal amino acid is at position 11. The NPM1c neoepitope is at position 12. In some embodiments, the C-terminal amino acid is at position 12, which is the length of the amino acid residue.

[0085] Neoepitopes derived from NPM1c that bind to HLA-A2 are known in the art. For example, Greiner (2012) Blood 120:1282 contains AIQDLCLAV(Sequence ID 1). ) and AIQDLCVAV (SEQ ID NO: 71), including 9mers that bind to HLA-A2. The amino acid sequence of the NPM1c neoepitope has been identified. As a further example, van der Lee (2019) J Clin Invest 129:774 includes CLAVEEVSL (SEQ ID NO: 72) The amino acid sequence of the NPM1c neoepitope that binds to HLA-A2 class I molecules, and further This includes VEEVSLRK (sequence number 73), AVEEVSLR (sequence number 74), AVE Includes EVSLRK (sequence number 75) and CLAVEEVSLRK (sequence number 76) NPM1 binds to MHC class I molecules encoded by other HLA haplotypes. We are identifying the amino acid sequence of the c neoepitope.

[0086] In some embodiments, MHC class I proteins (e.g., NPM1c:HLA-A2) and Mutant nucleophosmin proteins that form (or are presented by) a complex Antibodies that bind to antigens containing high-quality neoepitopes (e.g., specifically bind them) and An antigen-binding fragment in which a mutation occurs in the nucleophosmin protein Antibodies and their antigenic bonds are caused by a 4-nucleotide duplication in the gene encoding min. A composite fragment is provided herein. In some embodiments, MHC class I protein ( For example, NPM1c (HLA-A2) forms a complex with (or is presented by) (A cytoplasmic (located in the cytoplasm) mutant nucleophosmin protein neoepitol) An antibody that binds to an antigen containing (for example, specifically binds to) a p and its antigen-binding fragment, Provided herein. In some embodiments, the neoepitope is a variant nucleophore. It is a peptide of 8, 9, 10, 11, or 12 amino acids derived from the sumin protein. In some embodiments, the neoepitope is 10,1 of the mutant nucleophosmin protein. 5, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , derived from 85, 90, 95, or 100 amino acid residues, 8, 9, 10, 11, and It is a 12-amino acid peptide. In some embodiments, the neoepitope is a mutant nucleo 10, 15, 20, 25, 30, 35, 40, 45, 5 at the C-terminus of fosmin protein 0, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues A peptide consisting of 8, 9, 10, 11, or 12 amino acids derived from a base. Some embodiments So, the mutant nucleophosmin protein is the amino acid shown by SEQ ID NO: 56. Includes a sequence. In some embodiments, the mutant nucleophosmin protein is wild-type nucleo One or more mutations compared to the amino acid sequence of fosmin (e.g., SEQ ID NO: 54) For example, it includes an amino acid sequence having an insertion, deletion, or substitution. In some embodiments, a mutant nucleus The Ophosmin protein neoepitope is a protein containing the amino acid sequence of SEQ ID NO: 56. It is a peptide of 8, 9, 10, 11, or 12 amino acids derived from a substance. In some embodiments, The mutant nucleophosmin protein neoepitope is wild-type nucleophosmin One or more mutations (e.g., insertions) compared to the amino acid sequence of (e.g., SEQ ID NO: 54) 8, 9, 10, 11, derived from proteins containing amino acid sequences with (deletion, substitution) Alternatively, it is a 12-amino acid peptide. In some embodiments, it is a mutant nucleophosmintan. The protein neoepitope is the amino acid sequence 10, 15, 2 shown by SEQ ID NO: 56. 0, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 , 8, 9, 10, 11, or 12 amino acids derived from 90, 95, or 100 amino acid residues It is a peptide of nucleophosphate. In some embodiments, it is a mutant nucleophosmin protein neoe Pitope is compared to the amino acid sequence of wild-type nucleophosmin (e.g., SEQ ID NO: 54) A ta A peptide of 8, 9, 10, 11, or 12 amino acids derived from protein, one Or multiple mutations: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, Tans with a length of 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues Located within the protein region, a neoepitope is a protein region containing one or more mutations. It originates from the region. In some embodiments, the mutant nucleophosmin protein neoepitope is , the C-terminal 10, 15 of the protein having the amino acid sequence shown by SEQ ID NO: 56 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 8, 9, 10, 11, or 1 derived from 85, 90, 95, or 100 amino acid residues It is a two-amino acid peptide. In some embodiments, it is a mutant nucleophosmin protein. The oepitope has an amino acid sequence similar to that of wild-type nucleophosmin (e.g., SEQ ID NO: 54). In comparison, it has an amino acid sequence with one or more mutations (e.g., insertion, deletion, substitution). A peptide of 8, 9, 10, 11, or 12 amino acids derived from a protein, One or more mutations occur near the proximal end of the C-terminus (e.g., approximately 10, 15, 20, 25 from the C-terminus). , 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, Located within a protein region of 95 or 100 amino acid residues, the neoepitope is , derived from a region of protein containing one or more mutations. In some embodiments, MHC It formed a complex with a Las I protein (e.g., HLA-A2 protein) (or it The neoepitope of the protein containing the amino acid sequence of SEQ ID NO: 56 (presented by) An antibody or its antigen-binding fragment that specifically binds to an antigen is provided herein. It can be done.

[0087] In some embodiments, the mutant nucleophosmin protein has a C-terminal amino acid sequence MTD Includes QEAIQDLCLAVEEVSLRK (SEQ ID NO: 57). In some embodiments, MH It forms a complex with a Class I C protein (e.g., HLA-A2 protein) (or The C-terminal amino acid sequence presented therein is MTDQEAIQDLCLAVEEVSL Specific to antigens containing neoepitopes of the NPM1c protein, including RK (SEQ ID NO: 57). Antibodies that bind to and antigen-binding fragments thereof are provided herein. In some embodiments The neoepitope is the C-terminal amino acid sequence MTDQEAIQD of the NPM1c protein. 8, 9, 10, 11, or 12 derived from LCLAVEEVSLRK (Sequence ID 57) It is an amino acid peptide. In some embodiments, it is the HLA-A2 protein or HLA-A *02 Proteins encoded by allele groups (i.e., NPM1c:HLA) -A2) NPM1c neoepitone that forms a complex with (or is presented by) Antibodies that specifically bind to antigens containing p and antigen-binding fragments thereof are provided herein. In some cases, NPM1c is human NPM1c.

[0088] In some embodiments, MHC class I proteins (e.g., HLA-A2 protein or It formed a complex with the protein encoded by the HLA-A*02 allele group. (or the cytoplasmic mutant nucleophosmin protein neoe) presented therein Antibodies that bind to (e.g., specifically bind to) antigens containing pitopes, or cleavage of such antigens. It is a single piece, and the amino acid sequence of the neoepitope is AIQDLCLAV (SEQ ID NO: 1), A IQDLCVAV (sequence number 71), CLAVEEVSL (sequence number 72), VEEVS LRK (sequence number 73), AVEEVSLR (sequence number 74), AVEEVSLRK (distribution An antibody or its anti- Original binding fragments are provided herein. In some embodiments, HLA-A2 is used. The following are shown: AIQDLCLAV (sequence number 1), AIQDLCVAV (sequence number 71), C LAVEEVSL (sequence number 72), VEEVSLRK (sequence number 73), AVEEVS LR (sequence number 74), AVEEVSLRK (sequence number 75), and CLAVEEVSL An antibody that binds to an antigen containing an amino acid sequence selected from RK (SEQ ID NO: 76), and the same Antigen-binding fragments are provided herein. In some embodiments, HLA-A2 An antibody that binds to an antigen containing the presented amino acid sequence AIQDLCLAV (SEQ ID NO: 1) and its antigen-binding fragments are provided herein.

[0089] In some embodiments, the antibodies or antigen-binding fragments described herein are MHC class Control protein I alone and / or complexed with MHC class I protein Tide (for example, a control peptide has the same number of amino acids as a neoepitope, but neoe Does it not bind to proteins (which are derived from proteins different from the protein from which the pitope originated)? or not substantially combined.

[0090] In some embodiments, the antibodies or antigen-binding fragments described herein are cytoplasmic mutations. Type nucleophosmin protein neoepitope alone (MHC class I protein, for example) If HLA-A2 is not present, it does not bind, or does not bind substantially.

[0091] In some embodiments, the NPM1c neoepitope is the amino acid sequence AIQDLCLAV (distributed Column number 1) contains MHC class I proteins, HLA-A2 proteins (e.g., H It is a protein encoded by the LA-A*02:01 allele. In some embodiments, The NPM1c neoepitope is AIQDLCVAV (SEQ ID NO: 71), CLAVEEV SL (sequence number 72), VEEVSLRK (sequence number 73), AVEEVSLR (sequence number) (Sequence number 74), AVEEVSLRK (Sequence number 75) and CLAVEEVSLRK (Sequence number 74) The MHC class I protein contains an amino acid sequence selected from (76), and HLA-A 2 proteins (for example, proteins encoded by the HLA-A*02:01 allele) )

[0092] In some embodiments, it is complexed with Class I major histocompatibility complex (MHC class I) proteins. Includes a neoepitope containing the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) that formed the amino acid sequence. An antibody or its antigen-binding fragment that specifically binds to an antigen, wherein the amino acid sequence is AIQDL One, two, three, four, five, or six of the elements in CLAV (Sequence ID 1) Antibodies or antigen-binding fragments in which the amino acids are substituted are provided herein. In some embodiments, it is complexed with Class I major histocompatibility complex (MHC class I) proteins. Specifically for antigens containing neoepitopes including the amino acid sequence AIQDLCLAV that formed the amino acid sequence AIQDLCLAV The antigen to bind and its antigen-binding fragment, of which the amino acid sequence AIQDLCLAV One, two, three, or four of the amino acids are substituted in the antibody or its anti- Proto-binding fragments are provided herein. In some embodiments, amino acid substitutions are conservative. This is an amino acid substitution. In some embodiments, the amino acid substitution is in the AIQDLCLAV sequence (sequence number) This is a substitution with an amino acid residue of a similar size to the existing residue in (1). Some embodiments Therefore, amino acid substitutions are used to bind the antibodies and their antigen-binding fragments described herein to the antigen. It does not affect (or has virtually no effect on) the result.

[0093] In some embodiments, it is complexed with Class I major histocompatibility complex (MHC class I) proteins. Specifically for antigens containing neoepitopes including the amino acid sequence AIQDLCLAV that formed the amino acid sequence AIQDLCLAV A binding antibody and its antigen-binding fragment, wherein the amino acid sequence is AIQDLCLAV (sequence) One, two, or more of the anchor residues in number 1) are substituted (for example) , position 2 and / or position 9 of sequence number 1, for example, AIQDLCLAV(sequence number 1 The underlined residues of (), antibodies or their antigen-binding fragments are provided herein. In some embodiments, amino acid substitutions are used to break the binding of antibodies and their antigens as described herein. Binding to the antigen, or Class I major histocompatibility complex (MHC class I) protein ( For example, it does not affect (or substantially affects) the binding of neoepitopes to HLA-A2. (None). In some embodiments, the amino acid residue at position 2 of AIQDLCLAV (SEQ ID NO: 1) Group I is substituted with amino acid residue L (leucine). In some embodiments, AIQD The amino acid residue I at position 2 of LCLAV (Sequence ID 1) is amino acid residue V (valine). M (methionine), tyrosine (T), serine (S), glutamine (Q) or A (alool) It is replaced with (nin). In some embodiments, the part of AIQDLCLAV (sequence number 1) The amino acid residue V, located at position 9, consists of amino acid residues I (isoleucine), L (leucine), and M ( It is substituted with methionine or A (alanine).

[0094] In some embodiments, it is complexed with Class I major histocompatibility complex (MHC class I) proteins. Specifically for antigens containing neoepitopes including the amino acid sequence AIQDLCLAV that formed the amino acid sequence AIQDLCLAV A binding antibody and its antigen-binding fragment, wherein the amino acid sequence is AIQDLCLAV (sequence) One, two, three, four, five, or six of the amino acids in number 1) are substituted. The substitution is a conservative amino acid substitution, and the antibody or its antigen-binding fragment is specified herein. It is provided in which a complex with an MHC class I protein is formed. It specifically binds to antigens containing neoepitopes that include the amino acid sequences identified in Table 1. Antibodies and their antigen-binding fragments are provided herein.

[0095] [Table 1]

[0096] In some embodiments, this disclosure relates to Class I major histocompatibility complex (MHC class I) proteins. It specifically binds to the amino acid sequence AIQDLCLAV (SEQ ID NO: 1), which forms a complex with the substance. An antibody or antigen-binding fragment having the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) One, two, three, four, five, or six of these amino acids are substituted. The amino acid sequence in which an antibody or antigen-binding fragment forms a complex with an MHC class I protein. It has the same or substantially the same binding affinity to AIQDLCLAV (SEQ ID NO: 1) The present invention provides antibodies or antigen-binding fragments. In some embodiments, the present invention relates to Class I major tissue-compatible materials. The amino acid sequence AIQDLCL formed a complex with the MHC class I protein. An antibody that specifically binds to AV (SEQ ID NO: 1) and its antigen-binding fragment, wherein the amino acid One, two, three, four, or five of the elements in the sequence AIQDLCLAV (sequence number 1) Alternatively, six amino acids are substituted, and the antibody and its antigen-binding fragment are MHC class The amino acid sequence AIQDLCLAV (SEQ ID NO: 1) forms a complex with protein I. The antibody or its antigen-binding fragment, which binds specifically with the same or better affinity, is provided. Provided. In some embodiments, the antibodies and antigen-binding fragments described herein are Class Amino acid sequence A that forms a complex with major histocompatibility complex (MHC class I) proteins. It binds to IQDLCLAV (SEQ ID NO: 1), and the amino acid sequence AIQDLCLAV (SEQ ID NO: 1) 1) Any one, two, three, four, five, or six of the amino acids are substituted. Therefore, the antibody or antigen-binding fragment is 0.1-100 nM (for example, 0.1-50 nM, 0 K (0.1~25nM, 0.1~15nM) D It has. In some embodiments, this specification The antibodies and antigen-binding fragments described are Class I major histocompatibility complexes (MHC class I) The amino acid sequence AIQDLCLAV (Sequence ID 1) forms a complex with the protein. Combined, one, two, or three of the amino acid sequences AIQDLCLAV (SEQ ID NO: 1) Four, five, or six amino acids are substituted, and the antibody and its antigen-binding fragment are , less than 100 nM (for example, less than 50 nM, less than 25 nM, less than 15 nM, less than 7 nM, Less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, 0.9n Less than M, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM, 0.4 n K (less than M, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM) D They are joined together.

[0097] In some embodiments, MHC class I proteins, such as HLA-A2 (NPM1c:H LA-A2) binds to the NPM1c epitope, exhibiting anticancer or antitumor effects. It has an anti-cancer effect (for example, an in vivo anti-cancer effect, with optional selection, and the cancer is AML). Antibodies and antigen-binding fragments thereof are provided herein.

[0098] In some embodiments, this disclosure relates to Class I major histocompatibility complex (MHC class I) proteins. An antibody that specifically binds to an antigen containing an NPM1c neoepitope that has formed a complex with the substance, or the antigen-binding fragment, which includes a heavy chain variable region (VH) and a light chain variable region (VL). The present invention provides an antibody or an antigen-binding fragment thereof. In some embodiments, the neoepitope is AI Contains an amino acid sequence including QDLCLAV (SEQ ID NO: 1). In some embodiments, MHC clavicle The S1 protein is controlled by HLA-A alleles, including the HLA-A*02 allele group. In some embodiments, the HLA-A allele is HLA-A*02:01.

[0099] In some embodiments, the heavy chain variable region and / or light chain variable region described herein are included. (For example, having an array of heavy chain variable regions and / or light chain variable regions of YG1 scFV) (See, for example, the Sequence section and Examples) Anti-NPM1c:HLA-A2 antibody and Antigen-binding fragments are provided herein. In some embodiments, as described herein It has one or more complementarity determination regions (CDRs) (for example, C of YG1 scFv It has a DR. (See, for example, the Sequence section and Examples) Anti-NPM1c:HLA-A2 antibody and its antigen-binding fragments are provided herein. In some embodiments, NPM1c The antibody that binds to HLA-A2, or its antigen-binding fragment, is scFv. NPM1 The exemplary amino acid sequence of scFv, which specifically binds to c:HLA-A2, is shown in SEQ ID NO: 2. It is shown. In some embodiments, the amino acid sequence shown in SEQ ID NO: 2 is at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, less scFv having 98% or at least 99% amino acid sequence identity is specified herein. Provided in [location]. In some embodiments, the amino acid sequence shown in SEQ ID NO: 2 and at least 75%, 80%, at least 85%, at least 90%, at least 95%, at least scFv having 98% or at least 99% amino acid sequence identity, and sequence number At least 95% of the differences in identity with the amino acid sequence shown in No. 2 are due to the scFv's frame scFv located in the Mwork region (or not in the complementarity determination region (CDR)) is specified in this specification It is provided in [location].

[0100] In some embodiments, the heavy chain variable region (VH) of amino acid sequence SEQ ID NO: 5(YG1 scFv) Anti-NPM1c:HLA-A2 antibody containing VH (amino acid sequence of the same name) and its antigenicity Compound fragments are provided herein. In some embodiments, the amino shown in SEQ ID NO: 5 Acid sequence (amino acid sequence of the heavy chain variable region (VH) of YG1 scFv) and at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, less Anti-NPM1 containing VH having 98% or at least 99% amino acid sequence identity c:HLA-A2 antibodies and their antigen-binding fragments are provided herein. In one embodiment, the amino acid sequence shown in Sequence ID No. 5 is at least 75%, 80%, and at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% VH having % amino acid sequence identity, and the amino acid sequence shown in SEQ ID NO: 5 At least 95% of the differences in identity lie within the VH framework domain (or relative domain). VH (which is not in the complementarity determination region (CDR)) is provided herein.

[0101] In some embodiments, the light chain variable region (VL) of amino acid sequence sequence number 3 (YG1 scFv) Anti-NPM1c:HLA-A2 antibody containing VL having the amino acid sequence of the same name and its antigenicity Compound fragments are provided herein. In some embodiments, the amino shown in SEQ ID NO: 3 Acid sequence (amino acid sequence of the light chain variable region (VL) of YG1 scFv) and at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, less Anti-NPM1 containing VL having 98% or at least 99% amino acid sequence identity c:HLA-A2 antibodies and their antigen-binding fragments are provided herein. In this embodiment, the amino acid sequence shown in Sequence ID No. 3 is at least 75%, 80%, and at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% A VL having % amino acid sequence identity, and having the amino acid sequence shown in SEQ ID NO: 3 At least 95% or all of the differences in identity fall within the VL framework domain. A VL that is (or is not in the complementarity determination region (CDR)) is provided herein.

[0102] In some embodiments, the heavy chain variable region (VH) of amino acid sequence SEQ ID NO: 5(YG1 scFv) VH having the amino acid sequence of (YG1 scFv), and amino acid sequence number 3 (YG1 scFv) Anti-NPM1c:HLA-A2 anti- A body and its antigen-binding fragments are provided herein. In some embodiments, the sequence number The amino acid sequence shown in 5 (amino acid sequence of the heavy chain variable region (VH) of YG1 scFv) and at least 75%, at least 80%, at least 85%, at least 90%, less Both V have 95%, at least 98%, or at least 99% amino acid sequence identity. H, and the light chain variable region (VL) of the amino acid sequence (YG1 scFv) shown in Sequence ID No. 3. ) Amino acid sequence) and at least 75%, at least 80%, at least 85%, less Each contains 90%, at least 95%, at least 98%, or at least 99% amino acids. Anti-NPM1c:HLA-A2 antibodies containing VL with column identity and their antigen-binding fragments These are provided herein. In some embodiments, SEQ ID NO: 5 and SEQ ID NO: 3, respectively. The amino acid sequence shown and at least 75%, 80%, at least 85%, and at least 9 0%, at least 95%, at least 98%, or at least 99% amino acid sequence identity VH and VL having properties, and the amino acid combination shown in SEQ ID NO: 5 and SEQ ID NO: 3 At least 95% or all of the differences in identity with the column are VH and VL. VH and VL located in the Mwork region (or not in the Complementarity Determination Region (CDR)) Provided in the specification.

[0103] The CDRs of antibody or antigen-binding fragments in this disclosure are used in the art by Kabat, Ch. Defined in various ways, including othia, AbM, Contact, and IMGT. ru.

[0104] In some embodiments, the CDR of the antibody of this disclosure is defined according to the Kabat system. This is based on sequence variability (e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382). -391; Kabat EA et al, (1991) Sequences of Proteins of Immunological Interest, Fi fth Edition, US Department of Health and Human Services, NIH Publication No. 9 See 1-3242. The location of the Kabat CDR is determined according to methods known in the art. In one embodiment, the CDR of the antibody and its fragment described herein is Kabat systolic. Determined using the TEM. In some embodiments, determined using the Kabat system. , anti-NPM1c having one or more complementarity-determining regions (CDRs) of YG1 scFv HLA-A2 antibodies and their antigen-binding fragments are provided herein.

[0105] In some embodiments, the CDR of the antibody of this disclosure is defined according to the Chothia system. This is based on the location of the immunoglobulin structure loop region (e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927-948; Chothia C et al, (1992) J Mol Biol 227: 799-817; Tramontano A et al, (1 See 990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226. The term "Chothia CDR" and similar terms are recognized in the art. It was determined according to the method of Chothia and Lesk, 1987, J. Mol. Biol., 196:901-917. This refers to an antibody CDR sequence, which in this specification is referred to as "Chothia CDR" (e.g. For example, U.S. Patent No. 7,709,226 and Martin, A., "Protein Sequence and d Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kon Termann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (200 See 1). The location of the Chothia CDR is determined according to methods known in the art. In some embodiments, the CDR of the antibodies and their fragments described herein is Chot Determined using the hia system. In some embodiments, the Chothia system is used. The YG1 scFv has one or more complementarity determination regions (CDRs) as determined by An anti-NPM1c:HLA-A2 antibody and its antigen-binding fragment are provided herein. It can be done.

[0106] In some embodiments, the CDR of the antibody of this disclosure is defined according to the AbM system, which is AbM hypervariable region equivalent to a compromise between Kabat CDR and Chothia structural loops. Based on Oxford Molecular's AbM antibody modeling software, CDR is based on Oxford Molecular's AbM antibody modeling software. Using software (Oxford Molecular Group, Inc.) The position of the AbM CDR is determined according to methods known in the art. In this specification, the CDRs of antibodies and their fragments described herein are obtained using the AbM system. It is determined by the AbM system, YG1 scF Anti-NPM1c:HLA-A2 anti-v with one or more complementarity-determining regions (CDRs) A body and its antigen-binding fragments are provided herein.

[0107] In some embodiments, the CDR of the antibody of this disclosure is defined according to the IMGT system (I MGT (registered trademark), International ImMunoGeneTics i Information System (registered trademark) website imgt.org, founder and See director: Marie-Paule Lefranc, Montpellier, France; for example, Lefranc, M.- P., 1999, The Immunologist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic See Acids Res., 27:209-212. Both of these are incorporated herein by reference in their entirety. (It is included). The position of the IMGT CDR is determined according to methods known in the art. In one embodiment, the antibody and its fragment CDR described herein are used in the IMGT system. Determined using the IMGT system, YG 1 anti-NPM1c:HL with one or more complementarity determination regions (CDRs) of scFv A-A2 antibodies and their antigen-binding fragments are provided herein.

[0108] In some embodiments, the CDR of the antibody of this disclosure is defined according to the Contact system. The definition of Contact is based on the analysis of available complex crystal structures (bioinf.org.u k / abs) (See MacCallum RM et al., (1996) J Mol Biol 5: 732-745; for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in A ntibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Sprin See also ger-Verlag, Berlin (2001). The location of Contact CDR is in this technology. Determined according to methods known in the field. In one embodiment, the antibody and so described herein The CDR of the fragment is determined using the Contact system. In some embodiments, C One or more complementary YG1 scFvs, determined using the ontact system. Anti-NPM1c:HLA-A2 antibody with sex-determining region (CDR) and its antigen-binding fragment However, this is provided herein.

[0109] In some embodiments, it specifically binds to the NPM1c epitope presented by HLA-A2. In addition, one or two YG1 scFvs defined according to any of the above systems, Or three VH CD-Rs and / or one, two, or three VL CD-Rs Antibodies or fragments thereof containing are provided herein. For example, in one embodiment, H It specifically binds to the NPM1c epitope presented by LA-A2, and is activated by IMGT. As defined, one, two, or all three VH CDRs of YG1 scFv An antibody or fragment containing one, two, or all three VL CDRs , provided herein.

[0110] As is well known in the art, VH and VL are enclosed by a framework area. Contains a CDR (the CDR and FR sequences are in the following order in VH and VL) Appears: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4). Optional selection. In this case, the framework domain is the human framework domain.

[0111] In a particular embodiment, the heavy chain variable region (V) of amino acid sequence sequence number 5(YG1 scFv) Having one, two, or all three VH CDRs with the amino acid sequence of H) The anti-NPM1c:HLA-A2 antibody containing VH and its antigen-binding fragment are described herein. It is provided as follows. In some embodiments, the amino acid sequence number is defined by IMGT. Having one, two, or all three VH CDRs of heavy chain variable regions (VH) having 5 The anti-NPM1c:HLA-A2 antibody containing VH and its antigen-binding fragment are described herein. It will be provided.

[0112] In some embodiments, the light chain variable region (VL) of amino acid sequence sequence number 3 (YG1 scFv) VL having one, two, or all three VL CDRs having the amino acid sequence of VL An anti-NPM1c:HLA-A2 antibody containing L and its antigen-binding fragment are described herein. Provided. In some embodiments, amino acid sequence number 3 (as defined by IMGT) The amino acid sequence of the light chain variable region (VL) of YG1 scFv (one or two VLs) or anti-NPM1c:HLA-A2 antibody containing VLs that have all three VL CDRs Antigen-binding fragments thereof are provided herein.

[0113] In some embodiments, the heavy chain variable region (VH) of amino acid sequence SEQ ID NO: 5(YG1 scFv) VH having one, two, or three of all VH CDRs (amino acid sequence) H, and amino acids in the light chain variable region (VL) of amino acid sequence SEQ ID NO: 3(YG1 scFv) Includes one, two, or all three VL CDRs having an acid sequence. An anti-NPM1c:HLA-A2 antibody and its antigen-binding fragment are provided herein. ru.

[0114] In some embodiments, the VH CDR1 of amino acid sequence SEQ ID NO: 9, and the amino acid sequence SEQ ID NO: 1 It has VH CDR2 of 0 and / or VH CDR3 of amino acid sequence SEQ ID NO: 11 Anti-NPM1c:HLA-A2 antibody containing a heavy chain variable region (VH) and its antigen-binding fragment However, this is provided herein. In some embodiments, the VH of amino acid sequence SEQ ID NO: 9 CDR1, VH CDR2 of amino acid sequence number 10, and amino acid sequence number Anti-NPM1c:HLA-A2 containing heavy chain variable region (VH) with 11 VH CDR3 An antibody and its antigen-binding fragment, namely SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 Anti-NPM1c:HLA has one, two, three, four, or five amino acids substituted. -A2 antibodies and their antigen-binding fragments are provided herein. In some embodiments In some embodiments, amino acid substitutions are conservative substitutions. This is a substitution by an amino acid residue. In a particular embodiment, the amino acid substitution is as described above. It does not affect (or substantially does not affect) the binding of the antibody or antigen-binding fragment to the antigen as described in the details. (It has no effect), or does not improve.

[0115] In some embodiments, the light chain variable region (VL) CDR1 of amino acid sequence number 6, amino acid sequence VL CDR2 of SEQ ID NO: 7, and / or VL CDR3 of amino acid sequence SEQ ID NO: 8 The anti-NPM1c:HLA-A2 antibody containing VL and its antigen-binding fragment, as described in this specification, Provided in the book. In some embodiments, the light chain variable region (VL) of amino acid sequence number 6. )CDR1, VL CDR2 of amino acid sequence number 7, and / or amino acid sequence Anti-NPM1c:HLA-A2 antibody containing VL with VL CDR3 in column number 8 and An antigen-binding fragment of one, two, or three of SEQ ID NOs: 6, 7, or 8. Anti-NPM1c:HLA-A2 antibodies in which four or five amino acids are substituted. The antigen-binding fragment thereof is provided herein. In some embodiments, amino acid The substitution is a conservative substitution. In some embodiments, the amino acid substitution is an amino acid of similar size. This is a substitution by residue. In certain embodiments, amino acid substitutions are as described herein. It does not affect (or substantially affect) the binding of antibodies or antigen-binding fragments to the antigen. (To have no effect) or to improve.

[0116] In some embodiments, the heavy chain variable region (VH)CDR1 of amino acid sequence SEQ ID NO: 9, amino acid VH CDR2 of sequence SEQ ID NO: 10, and VH CDR3 of amino acid sequence SEQ ID NO: 11 VH having, and / or the light chain variable region (VL) CDR of amino acid sequence SEQ ID NO: 6 1. VL CDR2 of amino acid sequence SEQ ID NO: 7, and VL of amino acid sequence SEQ ID NO: 8 Anti-NPM1c:HLA-A2 antibody containing VL with CDR3 and its antigen-binding fragment This is provided herein. In a particular embodiment, the weight of amino acid sequence SEQ ID NO: 9 The chain variable region (VH) CDR1, the VH CDR2 of amino acid sequence SEQ ID NO: 10, and ami VH having VH CDR3 of amino acid sequence SEQ ID NO 11, and amino acid sequence SEQ ID NO 6 The light chain variable region (VL) CDR1 of the amino acid sequence SEQ ID NO: 7, and the VL CDR2 of the amino acid sequence SEQ ID NO: 7, and Anti-NPM1c:HLA-A2 containing VL with VL CDR3 of the minoic acid sequence SEQ ID NO: 8 Antibodies and their antigen-binding fragments are provided herein. In some embodiments, Anti-NPM1c:HLA-A2 antibodies including VH and VL as described in the specification and their anti- A protobinding fragment comprising one, two, three, four or VH and / or VL CDRs. An anti-NPM1c:HLA-A2 antibody with five amino acid substitutions and its antigen binding. Fragments are provided herein.

[0117] In some embodiments, the VH and / or VL regions of the antibody or fragment described herein In this case, one or more CDRs maintain specific binding to NPM1c:HLA-A2. As long as it is possible, one, two, three, four, or five amino acids may be different.

[0118] In some embodiments, the antibodies or fragments provided herein are affinity mature, that is, In addition, compared to the described antibody or fragment, in one or more complementarity-determining regions It has one or more modifications, and such one or more modifications are described in the antibody or This results in improved affinity of the antibody or fragment to the antigen compared to the fragment. In some embodiments, The antibodies or fragments provided herein are antigens (e.g., NPM1c:HLA-A2) ) compared to less than 100 nM (for example, less than 50 nM, less than 25 nM, less than 15 nM, 7 Less than nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM Less than 0.9 nM, less than 0.8 nM, less than 0.7 nM, less than 0.6 nM, less than 0.5 nM Kd (less than 0.4 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM) It possesses. In some embodiments, the antibody or fragment provided herein is an antigen (e.g., NP). For M1c (HLA-A2), less than 15nM, less than 10nM, less than 7nM, less than 5nM Full or less than 1 nM (e.g., 0.01 to 15 nM, 0.01 to 10 nM, 0.0 1 to 7 nM, 0.01 to 5 nM, 0.01 to 1 nM, 0.1 to 15 nM, 0.1 From 10nM, 0.1 to 7nM, 0.1 to 5nM, 0.1 to 1nM, 1 to 15n M, 1 to 10 nM, 1 to 7 nM, 1 to 5 nM, 5 to 15 nM, 5 to 10 nM, Alternatively, it has a Kd of 5 to 7 nM.

[0119] In some embodiments, the isolated antibody or its antigen-binding fragment is complemented by three light chain variable regions. Sex determination region (VL CDR 1-3) and three heavy chain variable region complementarity determination region (VH Includes CDR 1-3). In some embodiments, VH CDR1 is the one shown in Sequence ID No. 9. At least 80% sequence identity with the mino acid sequence, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or less At least 84% sequence identity, or at least 85% sequence identity, or at least 8 6% sequence identity, or at least 87% sequence identity, or at least 88% distribution Column identity, or at least 89% sequence identity, or at least 90% sequence identity. , or at least 91% sequence identity, or at least 92% sequence identity, At least 93% sequence identity, or at least 94% sequence identity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% Sequence identity, or at least 98% sequence identity, or at least 99% sequence identity It contains a monogeneous amino acid sequence. In some embodiments, VH CDR2 is in SEQ ID NO: 10 The amino acid sequence shown has at least 80% sequence identity, or at least 81% sequence identity. Identity, or at least 82% sequence identity, or at least 83% sequence identity, Or at least 84% sequence identity, or at least 85% sequence identity, or less At least 86% sequence identity, or at least 87% sequence identity, or at least 88% sequence identity, or at least 89% sequence identity, or at least 90% Sequence identity, or at least 91% sequence identity, or at least 92% sequence identity Sex, or at least 93% sequence identity, or at least 94% sequence identity, is at least 95% sequence identity, or at least 96% sequence identity, or less Both have 97% sequence identity, or at least 98% sequence identity, or at least 99% It contains an amino acid sequence having % sequence identity. In some embodiments, VH CDR3 is a sequence At least 80% sequence identity with the amino acid sequence shown in number 11, or at least 8 1% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity Column identity, or at least 84% sequence identity, or at least 85% sequence identity. , or at least 86% sequence identity, or at least 87% sequence identity, At least 88% sequence identity, or at least 89% sequence identity, or at least 90% sequence identity, or at least 91% sequence identity, or at least 92% Sequence identity, or at least 93% sequence identity, or at least 94% sequence identity Uniformity, or at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or less It contains amino acid sequences with at least 99% sequence identity.

[0120] In some embodiments, VL CDR1 has the amino acid sequence shown in SEQ ID NO: 6 and at least 80% sequence identity, or at least 81% sequence identity, or at least 82% Sequence identity, or at least 83% sequence identity, or at least 84% sequence identity Sex, or at least 85% sequence identity, or at least 86% sequence identity, is at least 87% sequence identity, or at least 88% sequence identity, or less Both have 89% sequence identity, or at least 90% sequence identity, or at least 91% % sequence identity, or at least 92% sequence identity, or at least 93% sequence identity Identity, or at least 94% sequence identity, or at least 95% sequence identity, Or at least 96% sequence identity, or at least 97% sequence identity, or less Amino acid combinations with at least 98% sequence identity, or at least 99% sequence identity. Includes a column. In some embodiments, VL CDR2 has an amino acid sequence similar to that shown in SEQ ID NO: 7. At least 80% sequence identity, or at least 81% sequence identity, or at least 82% sequence identity, or at least 83% sequence identity, or at least 84% Sequence identity, or at least 85% sequence identity, or at least 86% sequence identity Sex, or at least 87% sequence identity, or at least 88% sequence identity, is at least 89% sequence identity, or at least 90% sequence identity, or less Both have 91% sequence identity, or at least 92% sequence identity, or at least 93% % sequence identity, or at least 94% sequence identity, or at least 95% sequence identity Identity, or at least 96% sequence identity, or at least 97% sequence identity, Or having at least 98% sequence identity, or at least 99% sequence identity. It contains an amino acid sequence. In some embodiments, VL CDR3 is the amino acid shown in SEQ ID NO: 8. Sequence identity of at least 80%, or at least 81%, or less At least 82% sequence identity, or at least 83% sequence identity, or at least 84% sequence identity, or at least 85% sequence identity, or at least 86% Sequence identity, or at least 87% sequence identity, or at least 88% sequence identity Sex, or at least 89% sequence identity, or at least 90% sequence identity, is at least 91% sequence identity, or at least 92% sequence identity, or less Both have 93% sequence identity, or at least 94% sequence identity, or at least 95% % sequence identity, or at least 96% sequence identity, or at least 97% sequence identity Identity, or at least 98% sequence identity, or at least 99% sequence identity It contains the amino acid sequence it possesses.

[0121] In some embodiments, the antibody or antigen-binding fragment described herein is an isolated antibody It is a body or a fragment. In some embodiments, it is an antibody or antigen binding cleavage as described herein. The fragment is a purified antibody or fragment. In some embodiments, the antibody described herein is The antigen-binding fragment can then be detected by electrophoresis, for example, SDS-PAGE, isoelectric focusing. (by dynamic or capillary electrophoresis) or chromatography (e.g., ion exchange) Determined by reverse-phase HPLC, above 95%, above 97%, above 98% It is purified to a purity of over 99% (for example, Flatman, et al., J. Chromotogr See 848:79-87 (2007). In some embodiments, the antibodies or their anti- The primordial fragment is an isolated antibody or fragment that specifically binds to NPM1c:HLA-A2. In some embodiments, the antibody or antigen-binding fragment described herein is NPM 1c: A purified antibody or fragment that specifically binds to HLA-A2. In some embodiments, The NPM1c neoepitope is one of the ones described herein. In this case, the NP1M1c neoepitope is AIQDLCLAV (Sequence ID 1).

[0122] In some embodiments, the antibody or antigen-binding fragment described herein is an antigen (e.g., , at least 10 for NPM1c (MHC class I) -7 The binding affinity (Kd) of M It has. In certain embodiments, the antibody or antigen-binding fragment described herein is NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2) At least 10 -7M or stronger, at least 10 -8 M or stronger, less tomo10 -9 M or stronger, at least 500 nM or stronger, at 250 nM or stronger, at least 100 nM or stronger, at least 50 nM or stronger, at least 25nM or stronger, at least 20nM or stronger, at least 15 nM or stronger, or at least 10 nM It has a stronger binding affinity (Kd). In some embodiments, the antibody described herein The antigen-binding fragment is NPM1c:MHC class I antigen (for example, NPM1c:HL For A-A2), at least about 25 nM or stronger, at least about 15 nM or a stronger binding affinity (Kd) of at least about 10 nM or stronger. In some embodiments, the antibody or antigen-binding fragment described herein is NPM1 c: For MHC class I antigens (e.g., NPM1c:HLA-A2), 0.1 nM and 500nM, 0.1nM and 100nM, 0.5nM and 100nM, 0.1nM and 50nM , 0.5nM and 50nM, 0.1nM and 25nM, 0.5nM and 25nM, 0.1nM and 15nM, 0.5nM and 15nM, 0.1nM and 10nM, or 0.5nM and 10n Between M (or from the previous value to the next value), or from 1nM to 100nM (or between those values). It has a binding affinity (Kd) of any value. In some embodiments, the antibody described herein Alternatively, the antigen-binding fragment may be an NPM1c:MHC class I antigen (e.g., NPM1c:H For LA-A2), between approximately 0.1 nM and approximately 100 nM (or between the previous value and the later value) or has a binding affinity (Kd) of from about 0.5 nM to about 100 nM. In some embodiments , the antibodies or antigen-binding fragments thereof described herein have a binding affinity (Kd) of between about 0.1 nM and about 50 nM ( or from the former value to the latter value) or from about 0.5 nM to about 50 nM for an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2).

[0123] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Kon of at least 0.5 ± 0.02 × 10 Ms 4 -1 or higher for an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Kon of at least 1 ± 0.02 × 10 Ms 4 -1 or higher for an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Kon of at least 2.5 ± 0.02 × 10 Ms or higher for an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). 4 -1 In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Kon of at least 5 ± 0. 02 × 10 Ms 4 -1 or higher for an NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). In some embodiments, the antibodies or antigen-binding fragments thereof described herein have a Kon of at least 5 ± 0. For M1c (HLA-A2), 0.5±0.02×10 4 Ms -1 and 50±0.02 ×10 4 Ms -1 It has a Kon between (or from the previous value to the later value). In some embodiments The antibodies or antigen-binding fragments described herein are NPM1c:MHC class I For an antigen (e.g., NPM1c:HLA-A2), 1 ± 0.02 × 10 4 Ms -1 and 10±0.02×10 4 Ms -1 The Kon has a range between (or from the previous value to the next value). .

[0124] In some embodiments, the antibody or antigen-binding fragment described herein is NPM1c: For MHC class I antigens (e.g., NPM1c:HLA-A2), the result is 50±0.02× 10 -4 s -1 It has a Koff of less than . In some embodiments, the antibody described herein The antigen-binding fragment is NPM1c:MHC class I antigen (for example, NPM1c:HL For A-A2), 10±0.02×10 -4 s -1 It has a Koff of less than a certain amount. In some embodiments, the antibody or antigen-binding fragment described herein is NPM1c:MHC For the LA-I antigen (e.g., NPM1c:HLA-A2), 5 ± 0.02 × 10 -4 s -1 It has a Koff of less than . In some embodiments, the antibody or its antibody described herein The protobinding fragment is NPM1c:MHC class I antigen (e.g., NPM1c:HLA-A2). For comparison, 0.5±0.02×10 -4 s -1 and 50±0.02×10-4 s -1 During ( Or it has a Koff of (from the previous value to the later value). In some embodiments, as described herein The antibody or antigen-binding fragment is NPM1c:MHC class I antigen (for example, NPM For 1c (HLA-A2), between 1 ± 0.02 × 10 -4 s -1 and 15± 0.02 × 10 -4 s -1 It has a Koff between (or from the previous value to the later value).

[0125] In some embodiments, the antibodies described herein are monoclonal antibodies or their antigenic antibodies. It is a composite fragment. In some embodiments, the antibodies described herein are humanized antibodies or human antibodies. It is a body. In some embodiments, a human antibody or an antigen-binding fragment of a human antibody is used herein. Provided as follows. In some embodiments, a humanized antibody or an antigen-binding fragment of a humanized antibody is provided as described in this specification. Provided in the book. In some embodiments, a chimeric antibody or an antigen-binding fragment of a chimeric antibody ( Chimeric antibodies are antibodies that have a variable region of one species and a constant region of another species. Provided in the specification.

[0126] The antibodies provided herein are particularly effective against antigens (e.g., NPM1c:HLA-A2). Heterogenetically binding immunoglobulin molecules, and the same or substantially the same antigen as the antibody. Examples include immunoactive fragments of such molecules that bind to a link. In some embodiments, antibodies Or, the antigen bound by that antigen-binding fragment is MHC class on the surface of cancer cells. It is presented by molecule I (e.g., HLA-A2). In some embodiments, cancer cells These are AML cells.

[0127] In some embodiments, the anti-NPM1c:HLA-A2 antibody is one of the three V antibodies described herein. H CDR and / or three VL CDRs, human or human-derived framework regions A human antibody or humanized antibody or a constant region including a human or human-derived constant region These are immunoglobulins. Non-limiting examples in the human framework domain are described in this art. For example, Kabat et al. (1991) Sequences of Proteins of Immunological Int erest Fifth Edition, US Department of Health and Human Services, NIH Publicati on No. 91-3242; Sims et al. J. Immunol. 151 :2296 (1993); Carter et al. Proc. Na tl. Acad. Sci. USA, 89:4285 (1992); Presta et al. J. Immunol., 151 :2623 (1993); Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008); Baca et al., J. Biol Chem. 272: 10678-10684 (1997); Rosok et al., J. Biol. Chem. 271 :22611-22618 ( See Chothia et al., J. Mol. Biol. 278:457-479 (1998). Preferably, To improve the binding of the antibody described in the document to NPM1c:HLA-A2, one or Multiple amino acid substitutions can be added to the framework region.

[0128] In some embodiments where the antibody is an immunoglobulin, the type of antibody that can be used is IgG. These include IgE, IgM, IgD, IgA, and IgY, but the range is not limited to these. The classes of antibodies that can be used include IgG1, IgG2, IgG3, IgG4, IgA1, I gA2 is one example, but is not limited to these. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody or an IgG4 antibody. The antibody contains the wild-type IgG1 heavy chain constant region. In some embodiments, the antibody contains the wild-type Ig It includes a G4 heavy chain constant region. In some embodiments, the antibody includes a mutant IgG1 heavy chain constant region. In some embodiments, the antibody contains a variant IgG quadruplex constant region. The type IgG4 heavy chain constant region is substituted with EU numbering S228P, L235E, L235 A, or any one of a combination thereof. In some embodiments, the antibody is small It contains at least one Fc domain with a mutation.

[0129] In some embodiments, single-chain antibodies, such as single-chain Fv(scFv), are provided herein. In some embodiments, scFv is human scFv or humanized scFv. In some embodiments, scFv includes a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker is a Gly-Ser linker. In this case, the Gly-Ser linker is (Gly4Ser)1 (Sequence ID 58), (Gly (4Ser)2 (SEQ ID NO: 59), (Gly4Ser)3 (SEQ ID NO: 60), and (Gl Selected from the group consisting of y4Ser)4 (Sequence No. 61). In some embodiments, Gly- The Ser linker contains the amino acid sequence SGSSGGSSSG (SEQ ID NO: 4). In this context, the antigen-binding fragment of the antibody is not limited to Fv fragments, Fab fragments, etc. , F(ab') fragment, F(ab')² fragment, or disulfide-linked Fv(sdFv) Possible antigen-binding fragments of antibodies are provided herein. In one embodiment, Fv Fragments are provided herein. In one embodiment, a Fab fragment is provided herein. Provided. In one embodiment, fragment F(ab') is provided herein. In this context, the F(ab')2 fragment is provided herein.

[0130] In some embodiments, the antibody or antigen-binding fragment described herein is such an antibody It can induce cytotoxicity against cancer cells targeted by the body or fragments, Cell damage is caused by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADC), and antibody-dependent cell-mediated cytotoxicity (ADCC). P), complement-dependent cell injury (CDC), or toxins conjugated to such antibodies or fragments. This may be due to the cytotoxicity of the element or drug. In some embodiments, as specified herein The antibodies or antigen-binding fragments described herein contain ADCC, ADCP, and CDC. In some embodiments, the antibody or antigen-binding fragment described herein is ADCC and It has ADCP. In some embodiments, the antibody or its antigen binding cleavage as described herein. The fragments possess either ADCC activity only or CDC activity only.

[0131] In one embodiment, the antibody or antigen-binding fragment described herein is an antibody-dependent antigen. Mediates cell-mediated cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP) Methods for producing antibodies having ADCC and / or ADCP functions are described in the Art of this Technology. This is publicly known. Generally, the Fc region of an antibody is associated with neutrophils and macrophages that lead to ADCC. , its binding to Fc receptors on NK cells, eosinophils and mast cells, and to ADCP It mediates the binding of Fc receptors to connected macrophages, neutrophils, and dendritic cells. In some embodiments, the antibody has ADCC activity, and the Fc region of the antibody is human IgG Antibodies of the IgE type are provided herein. In one embodiment, it is assumed that The Fc region of the antibody is of the IgG1 isotype. In one embodiment, it is assumed that The Fc region of the antibody is of the IgG2 isotype. In one embodiment, The Fc region of the antibody is of the IgG3 isotype. The antibody has its ADCC and / Alternatively, it can be bioengineered to increase ADCP activity (e.g., mutations) (by crosslinking, disulfide bond formation, or oligosaccharide addition) (e.g., Natsume et al.) See Drug Des Devel Ther. 3:7-16, 2009. This is incorporated herein by reference. (In one embodiment, the CH2 and / or CH3 domains of the Fc region of the antibody are AD To improve CC and / or ADCP activity, fucose residues are reduced or removed. To remove them, they are modified at the glycosylation sites (e.g., Liu et al., 2015, Ca Immunol. Res. 3:173-183; Satoh et al., 2006, Expert Opin Biol. Ther. 6:1161-117 See 3; both of these are incorporated herein by reference). In one embodiment, human The Fc region of the IgG1 isotype is alanine substituted at position 333 of the CH2 domain. It is mutated. In one embodiment, the Fc region of the human IgG1 isotype is mutated by the following residues. Mutations: S239D, I332E, and A330L (e.g., Lazar et al., 20 See 06, PNAS 103:4005-4010 (which is incorporated herein by reference). One implementation In this state, the Fc region of the human IgG1 isotype is mutated to the following residue: S239 D, I332E, and G236A (e.g., Richards et al., 2008, Mol. Cancer The (See r. 7:2517-27). In one embodiment, the Fc region is located at positions 298, 333 and / or It contains an amino acid substitution at 334 (EU number), which improves ADCC activity.

[0132] In one embodiment, the antibody or antigen-binding fragment described herein is a complement-dependent molecule. It mediates cell damage (CDC). Methods for producing antibodies with CDC function are known in the art. In some embodiments, the antibody has CDC activity, and the Fc region of the antibody is human Ig Antibodies of type G or IgM are provided herein. In one embodiment, The Fc region of the antibody being determined is that of the IgG1 isotype. In one embodiment, The Fc region of the antibody being tested is of the IgG2 isotype. In one embodiment, it is assumed that The Fc region of the antibody is of the IgG3 isotype. The antibody's CDC activity is It can be bioengineered to increase (e.g., by mutating) (e.g., Moore et al., 2010, MAbs 2(2):181-189; Idusogie et al., 2001, J Immunol. 166(4): See 2571-5; Natsume et al., 2009, Drug Des Devel Ther. 3:7-16; all of these (as incorporated herein by reference). In one embodiment, an antibody having IgG Fc is used. In that Fc region, the N-glycan structure at the glycosylation site is fucose and cyanoglycan. It transforms into a G0 glycan type that terminates with N-acetylglucosamine without an alic acid residue. Sea urchins are manipulated biotechnically. In one embodiment, for example, U.S. Patent No. 6,194,551 Specification, International Publication No. 99 / 51642, or Idusogie et al., J. Immu As described in nol. 164:4178-4184 (2000), to improve CDC activity, F Modify region c.

[0133] In one embodiment, the antibody or antigen-binding fragment described herein is a cytotoxic agent. It binds to an agent (e.g., a toxin or drug). Cytotoxic agents induce cell death, Alternatively, they may be drugs that inhibit important cellular functions. Cytotoxic drugs include chemotherapeutic agents and proliferation agents. This may be, but is not limited to, an inhibitor, a radioisotope, or a toxin. In one embodiment, The antibodies or antigen-binding fragments described herein are toxins (e.g., diphtheria A chain, Exotoxin A chain, lysine A chain, abrin A chain, modesin A chain, dianthin protein, bitter Momordica charantia inhibitors, Crotin, Geronin, Neomycin Syn, Trichothecene, Phenomycin, Mitogerin, Restrictosin, Soapwort (s Apaonaria officinalis inhibitors, curcin, pokeweed (Phytolaca Americana) protein, Chinese tung tree (Aleur) Ites fordii) protein, or α-salsin) is bound to (for example, con (to be jugated). In one embodiment, the antibody or antigen-binding fragment described herein is , radioactive isotopes (e.g., P 32 , I 131 , I 125 At 211 , Y 90 Re 1 86 Re 188 Sm 153 , Bi 212 , or Pb 212 ) is combined (for example (conjugated). In one embodiment, the antibody or antigen binding described herein. The fragments are drugs (e.g., antimetabolites, folate antagonists, anthracyclines (e.g., doxo) Rubicin), methotrexate, taxane (e.g., docetaxel), paclitaxel, (bound to auristatin, dorastatin, meitansinoid, or calitiamycin) To be (for example, to be conjugated). Methods for producing antibody-drug conjugates are: Drugs and antibodies that are known in the art (and can be used as such conjugates) (Including linkers that can be used to connect objects) (e.g., Peters & Brown, 2015, Bios) (See ci. Rep. 35, e00225, doi:10.1042 / BSR20150089).

[0134] Also, antibodies or fragments thereof that bind to the same epitope, and / or human NMP1 c: Regarding binding to HLA-A2, any of the antibodies and fragments described herein ( For example, it includes the VH and VL amino acid sequences shown by SEQ ID NOs. 5 and 3, respectively. Antibodies or fragments thereof that compete with (antibodies or antibody fragments) are also included in the scope of this disclosure. Antibodies and their fragments that recognize or compete for binding to the same epitope are It can be identified using conventional methods. One such method is, for example, An immunoassay is an immunoassay that shows the ability of an antibody to block the binding of another antibody to a target antigen. One example is competitive binding assays. In competitive binding, the test antibody and the common antigen, for example, NM P1c: Determined in an assay that inhibits the specific binding of a reference antibody to HLA-A2. Numerous types of competitive binding assays are known, for example, solid-phase direct or indirect radiopharmaceutical assays. RIA (Rich Immunoassay), Solid-Phase Direct or Indirect Enzyme Immunoassay (EIA), Sun Deutsch competition assay (see Stahli et al., Methods in Enzymology 9:242 (1983)) Solid-phase direct biotin-avidin EIA (Kirkland et al., J. Immunol. 137:3614 (1986)) See also: Solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (Harlow and Lan e. See Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988); I Solid-phase direct labeling RIA using -125 labeling (Morel et al., Mol. Immunol. 25(1):7 (1 See 988); Solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546) (1990)); and directly labeled RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (19 90)) is an example. Typically, such assays involve purified antibodies bound to a solid surface. Cells containing the progenitor (e.g., NPM1c:HLA-A2) or either of these, unlabeled. Test antibody and labeled reference antibody (e.g., V, indicated by SEQ ID NOs. 5 and 3, respectively) This involves the use of antibodies containing H and VL amino acid sequences. Competitive inhibition occurs in the presence of the test antibody. It is measured by measuring the amount of label bound to a solid surface or cell. Typically, the test Antibodies are present in excess. Typically, when there is an excess of competing antibodies, it is because they are common antibodies to the reference antibody. Specific binding to the source is at least 50-55%, 55-60%, 60-65%, and 65-70%. Inhibits by %, 70-75%, or more. Other methods include, for example, epitometra. Pmapping methods, for example, provide atomic resolution for the crystals of antigen:antibody complexes that provide epitope resolution. X-ray analysis is one example. Other methods involve the use of antibodies against antigen fragments or mutated variants of the antigen. Binding is monitored, and a decrease in binding due to alteration of amino acid residues within the antigen sequence indicates an epitope. It is often considered an indicator of constituent elements. In addition, it is used for epitope mapping. Computer-based combination methods can also be used. These methods allow the target antibody to be obtained , specific short peptides, combinatorial phage display peptide library - It relies on the ability to affinity-separate from. Then, these peptides are peper Determination of epitopes corresponding to antibodies used to screen a cytolibrary. It is considered a lead for the sake of justice. Regarding epitope mapping, it is discontinuous in three dimensions. Computational algorithms have also been developed that have been shown to map epitopes.

[0135] Methods for identifying neoepitopes In some embodiments, the present disclosure describes how the present disclosure forms a complex with an MHC molecule (or thereby An antibody that specifically binds to an antigen containing a neoepitope (presented), or an antibody that cleaves its antigen binding. Provides a piece. In some embodiments, the neoepitope is tumor-specific or cancer-specific. It is an oepitope. In some embodiments, the MHC molecule is an MHC class I molecule.

[0136] The tumor-specific antigen or cancer-specific antigen from which the neoepitope originated is a non-silent cell. It contains altered amino acid sequences resulting from cytoplasmic mutations. For example, neoepitope derived from mutations. A point mutation (for example, a non-synonymous mutation that results in a different amino acid in a protein); a stop code The nucleotide is modified or deleted, resulting in a longer nucleotide having a novel tumor-specific sequence at the C-terminus. Read-through mutations that result in protein translation; resulting in the inclusion of introns in mature mRNA. Therefore, splice site mutations that result in unique tumor-specific protein sequences; two types Chromosomal rearrangement that produces chimeric proteins with tumor-specific sequences at the junction of proteins (i.e., gene fusion); novel open-source proteins with novel tumor-specific protein sequences. Frameshift mutations or deletions that result in a leading frame; and those caused by translocations. ru.

[0137] Methods for identifying tumor neoepitopes caused by tumor-specific or cancer-specific mutations , which is publicly known in the art (e.g., Richters, et al. (2019) Genome Medicine 11:56; See Liu, et al (2017) Cell 168:600). Such methods generally involve tumor-specific Identification of heterologous mutations (e.g., using deep sequencing methods for nucleic acids or proteins) Identification of the patient's human leukocyte antigen type and the corresponding major histocompatibility complex present in the tumor. Prediction, identification of neoepitope (e.g., potentially binding to the patient's HLA allele), To generate a set of candidate T cell epitopes based on mutations present in tumors, validation is performed. (By applying a peptide-MHC binding prediction algorithm or analytical method, In voluntary selection, demonstration of antigen-specific T cells against selected neoepitopes, or candidate neoepitopes. One example is the demonstration that the epitope is bound to HLA proteins on the tumor surface.

[0138] Deep nucleic acid methods are well known in this art. Any suitable sequence analysis method is used. One such method is, for example, based on chain termination sequencing. One example is sequence analysis using conventional Sanger sequencing (for example, Sanger See er, et al (1977) PNAS 74:5463). As a further example, as a method of sequence analysis... One example is the use of next-generation sequencing (NGS). NGS methods are used in the art. It is publicly known, and sequencing techniques based on pyrosequencing, and synthesis of Illum ina HiSeq and MiSeq sequencing, supported ol igonucleotide ligation and detection(SOL iD), DNA nanoball sequencing, Ion Torrent sequencing Single-molecule real-time sequencing (SMRT), Helicos sequencing Examples include , and Nanopore sequencing.

[0139] Nucleic acid sequencing involves sequencing the entire tumor genome, tumor exomes (protein-coding D2). NA), targeted regions of the genome (e.g., HLA loci) or tumor transcripts This is performed on a known control set. In some embodiments, the sequencing results are compared with a known control set. or sequencing performed on normal tissue or the corresponding normal tissue of the patient It is compared with analysis. One or more algorithms are used to analyze the sequencing data. Identify different classes of existing somatic mutations. For example, in some embodiments, the algorithm The SM is used to detect the diversity caused by single nucleotide variants (for example, Co rnish, et al (2015) Biomed Res Int 2015:456479; Ghoneim, et al (2014) BMC Res No See tes 7:864; Kroigard, et al (2016) PLoS One 11:e0151664), and / or i Used to detect the diversity that arises from the 'ndel' (e.g., Mose, et al (2014)). See 30:2813-2815; Narzisi, et al (2014) Nat Methods 11:1033-1036). Furthermore, In some embodiments, the detection of the fusion of two protein-coding sequences is performed using RNA sequencing. This is done by analyzing sequencing data and / or whole-genome sequencing data (e.g.) For example, Li, et al (2011) Bioinformatics 27:1708; Scolnick, et al (2015) PLoS One 1 0:30128916; Zhang, et al (2016) Genome Res 26:108; Kumar, et al (2016) Wiley Int (See erdiscip Rev RNA 7:811). A variant in tumor DNA or RNA is present. When detected, the effect of each variant on the amino acid sequence of the translated polypeptide is... , determined using computer calculation tools known in the art. Furthermore, translated Prediction of cleavage sites in polypeptides and MHC class I antigen processing Several tools are available to help identify peptides. Such tools are not limited in their scope. Examples include NetChop20S, NetChopCterm, and Protea SMM can be cited (for example, Nielsen, et al (2005) Immunogenetics 57:33; Tenzer, See et al (2005) Cell Mol Life Sci 62:1025.

[0140] Protein sequencing methods are also known in the art. In some embodiments, Protein sequencing is performed on the tumor proteome. In some embodiments, protein Using chromatographs, the presence of mutant peptides bound to MHC proteins on tumor cells was determined. Identify or validate the peptide from tumor cells or immunoprecipitated HL from tumors. It is eluted from molecule A with acid and then identified using mass spectrometry.

[0141] In some embodiments, once a variant tumor-specific peptide is identified, the MHC class Predicting its binding to molecule I or II requires knowledge of the patient's HLA haplotype. Human MHC is coded by the HLA gene complex located on chromosome 6p21.3. This locus is highly polymorphic, with over 12,000 alleles identified. Because HLA genes are individualized, accurate HLA haplotype determination is necessary. Methods for determining LA haplotypes are known in the art. For example, HLA haplotype determination The determination is based on sequence-specific PCR amplification and Sanger sequencing or NGS. This is done using sequencing methods. Based on sequencing data. Identifying HLA class I and II haplogroups involves multiple algorithms, for example, Po lysolver(Shukla, et al (2015) Nat Biotech 33:1152), HLAMiner( Warren, et al (2012) Genome Med 4:95), and OptiType (Szolek, et al (2 014) Bioinformatics 30:3310) is available.

[0142] In some embodiments, the computer algorithm estimates the neoepitope, i.e. , in the form of a peptide-presenting complex, it is bound by a class I or class II MHC molecule. Next, we predicted the peptide sequence recognized by the T cell receptor on T lymphocytes in this form. It is used for identifying peptides that bind to MHC class I. A non-limiting example is SMM (Nielsen, et al (2007) BMC Bioinformatics 8:238) , SMMPMBEC (Kim et al (2009) BMC Bioinformatics 10:394), Pickpoc ket (Zhang, et al 2009) Bioinformatics 25:1293), NetMHC (Andreatta, et al. al 2016) Bioinformatics 32:511), NetMHCpan (Jurtz et al (2017) J Immu nol 199:3360), NetMHCcons (Karosiene, et al (2012) Immunogenetics 64: 177), MHCflurry (O'Donnell, et al (2018) Cell Syst 7:129), and E One example is DGE (Bulik-Sullivan et al (2018) Nat Biotech).

[0143] In some embodiments, once the presumed neoepitopes are selected, they are in Further testing is conducted using in vitro and / or in vivo assays. In this embodiment, selected peptides are synthesized and encoded by different HLA haplotypes. To determine the binding to the MHC molecule, screening is performed using a human HLA panel. do.

[0144] Method for producing antibodies The antibodies and fragments described herein may be prepared by any method known in the art. It is possible.

[0145] In some embodiments, the methods described herein are, for example, phage display techniques. Techniques, bacterial displays, yeast surface displays, eukaryotic virus displays, mammalian movements Physical cell display and cell-free (e.g., ribosome display) antibody screen This can include techniques such as (e.g., Etz et al. (2001) J Bacteriol 183:6924-6935; Cornelis (2000) Curr Opin Biotechnol 11:450-454; Klemm et al. (2000) Microbiolo gy 146:3025-3032; Kieke et al. (1997) Protein Eng 10:1303-1310; Yeung et al. (20 02) Biotechnol Prog 18:212-220; Boder et al. (2000) Methods Enzymology 328:430-4 44; Grabherr et al. (2001) Comb Chem High Throughput Screen 4:185-192; Michael e (1995) Gene Ther 2:660-668; Pereboev et al. (2001) J Virol 75:7107-7113; chaffitzel et al. (1999) J Immunol Methods 231:119-135; Chao et al., 2006, Natur e Protocols 1(2):755-768; and Hanes et al. (2000) Nat Biotechnol 18:1287-1292 reference).

[0146] Methods for identifying antibodies using various phage display methods are available in the art. It is publicly known. In phage display, the functional antibody domain is the port that encodes it. It is presented on the surface of phage particles that possess a renucleotide sequence. Using this, a repertoire or combinatorial antibody library (e.g., human or The antigen-binding domain of an antibody expressed from a mouse, for example, Fab, Fv, or dys Rufid-binding stabilized Fv antibody fragments can be presented in these methods. Phages are typically filamentous phages, such as fd and M13. Antigen binding The domain is one of the phage coat proteins pIII, pVIII, or pIX. It is expressed as a recombinantly fused protein. For example, Shi et al. (2010) JMB 39 See 7:385-396. To manufacture the immunoglobulins or fragments thereof described herein. An example of a phage display method that can be used is Brinkman et al. (1995) J Immun. ol Methods 182:41-50; Ames et al. (1995) J Immunol Methods 184:177-186; rough et al. (1994) Eur J Immunol 24:952-958; Persic et al. (1997) Gene 187:9-18 ; Burton et al. (1994) Advances in Immunology 57:191-280; and PCT Open International Public release pamphlet No. 90 / 02809, International release pamphlet No. 91 / 10737, International Publication No. 92 / 01047 pamphlet, International Publication No. 92 / 18619 pamphlet International publication pamphlet No. 93 / 11236, International publication pamphlet No. 95 / 15982 Examples include those disclosed in Lett and International Publication No. 95 / 20401. A preferred method is, for example, U.S. Patent No. 5,698,426; and Article 5, 22 Specification No. 3,409; Specification No. 5,403,484; Specification No. 5,580,717 Specification No. 5,427,908; Specification No. 5,750,753; No. 5,82 Specification No. 1,047; Specification No. 5,571,698; Specification No. 5,427,908 Specification No. 5,516,637; Specification No. 5,780,225; No. 5,65 Specification No. 8,727; Specifications No. 5,733,743 and No. 5,969,108 It is also stated in the detailed document.

[0147] In some embodiments, the phage display antibody library is used to immunize phage-displayed It can be produced using mRNA collected from B cells of mammals. For example, Spleen cell samples containing B cells are immunized by the NPM1c:HLA-A2 complex as described above. It can be isolated from treated mice. mRNA is isolated from cells and processed according to standard It can be converted to cDNA using molecular biological techniques. For example, Sambrook et al. (1989) “Molecular Cloning: A Laboratory Manual, 2nd Edition,” Cold Spring Ha rbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane (1988), supra; B See enny KC Lo (2004), above; and Borrebaek (1995), above. Immunoglobulins Using cDNA encoding the variable regions of heavy and light chain polypeptides, To construct a phage display library. For example, see Merz et al. (1995) J Neurosci Methods 62(1-2):213-9; Di Niro e t al. (2005) Biochem J 388(Pt 3):889-894; and Engberg et al. (1995) Methods Mol This is described in Biol 51:355-376.

[0148] Methods for identifying antibodies using yeast surface display are well known in the art. Yes, there is yeast that can be used to produce the antibodies and fragments described herein. An example of a surface display method is described in Chao et al., 2006, Nature Protocols 1(2):755-768. Including the methods listed.

[0149] In some embodiments, the method for producing antibodies described herein is used for the target (e.g., non- The procedure may include the step of immunizing a human mammal with a suitable immunogen. Suitable immunogens for producing any of the antibodies described herein are as follows: For example, to produce an antibody that binds to NPM1c:HLA-A2, a person skilled in the art can, Suitable subjects (e.g., non-human mammals, e.g., rats, mice, gerbils, hamsters) - Dogs, cats, pigs, goats, llamas, horses, or non-human primates) for example, NPM1 When the neoepitope is AIQDLCLAV (SEQ ID NO: 1), NPM1c:HL Immunotherapy with antigens containing the A-A2 complex can be performed. Suitable subjects (e.g., non- In human mammals, a sufficient number of subsequent follow-ups to induce antibody production by that mammal. Immunotherapy can be performed with a suitable antigen, along with additive immunotherapy. The immunogen is an adjuvant. It can be administered to the target (e.g., non-human mammals) along with the band.

[0150] Methods for producing antibodies using hybridoma technology are well known in this field. In this embodiment, the method involves a hybrid that secretes a monoclonal antibody that binds to an immunogen. The process includes the step of preparing a suitable mammalian cell line, for example, a laboratory mouse. Immunotherapy using the NPM1c:HLA-A2 complex described above is performed. Antibody-producing cells in mammals (e.g., B cells of the spleen) undergo at least one follow-up of immunogens. They can be isolated 2 to 4 days after immunoassay and then briefly grown in culture. After that, fusion is performed with cells from a suitable myeloma cell line. The cells are, for example, fused with a fusion promoter, e.g. For example, fusion can be performed in the presence of vaccinia virus or polyethylene glycol. It is possible. Cloning the hybrid cells obtained through fusion will produce cells that secrete the desired antibody. Select a clone. For example, a Balb / c mouse that has been immunized with a suitable immunogen. The spleen cells were fused with cells from the myeloma cell line PAI or the myeloma cell line Sp2 / 0-Ag 14. They can be combined. After fusion, the cells can be combined into normal myeloma cells and the desired hybridoma cells. To prevent overgrowth of cells, a selective medium, such as HAT medium, is added at regular intervals. The cells are then grown in a suitable culture medium. Next, the resulting hybrid cells are subjected to the desired antibody (for example... Next, we screen for the secretion of NPM1c (an antibody that binds to HLA-A2).

[0151] In some embodiments, those skilled in the art can, for example, refer to U.S. Patent No. 6,300,064 (Kn appik et al. (for Morphosys AG) and Schoonbroodt et al. (2005) Nucleic Aci As described in ds Res 33(9):e81, the target antibody from a non-immune-based library It is possible to identify them.

[0152] In some embodiments, a combination of selection and screening is used, for example, for hybrids From a population of antibodies derived from phages or from a phage display antibody library, select the target antibody. It can be determined. Preferred methods are known in the art, for example, Hoogenboom (1997) ) Trends in Biotechnology 15:62-70; Brinkman et al. (1995), supra; Ames et al. (1 995), above; Kettleborough et al. (1994), above; Persic et al. (1997), above; and Burton et al. (1994), as described above. For example, each is a bacteriopha phageocoat proteins (e.g., pIII, pVIII, or pIX of M13 phage) Multiple phagemide vectors encoding fusion proteins with different antigen-binding regions, Prepared using standard molecular biological techniques, then bacteria (e.g., Escherichia coli (E. coli)) Introduce it into the population of li). Bacteriophage expression in bacteria is, in some embodiments Therefore, the use of helper phages may be necessary. In some embodiments, helper phages - Phages are not required (e.g., Chasteen et al., (2006) Nucleic Acids Res 34 (See (21):e145). Phages produced from bacteria are recovered and then, for example, solid support The phage is brought into contact with the target antigen that is bound to (immobilized) the body. The composite can be brought into contact with the solid support, and then the composite is bonded to the solid support.

[0153] A subset of antibodies screened using the above method is subjected to a method known in the art. Using immunological or biochemical methods, a specific antigen (e.g., NPM1c:H) is identified. Their specificity and binding affinity to LA-A2 can be characterized. For example, the specific binding of antibodies to NPM1c:HLA-A2 is investigated using immunology or biochemistry. Methods based on, for example, but not limited to, ELISA assays and SPR assays as described above. We use immunoprecipitation assays, affinity chromatography, and equilibrium dialysis. This can be determined by using this method to analyze the immunospecific binding and cross-reactivity of antibodies. Suitable immunoassays include Western blotting, RIA, and ELISA (enzyme-conjugated). Immunoadsorption assay, "sandwich" immunoassay, immunoprecipitation assay, immunodiffusion assay Assay, agglutination assay, complement fixation assay, immunoradioquantification assay, fluorescence immunoassay , and competitive and non-competitive assays using methods such as protein A immunoassays. Examples include, but are not limited to, stems. Such assays are conventional and It is well known in the technical field.

[0154] Methods for producing chimeric antibodies are well known in the art (e.g., Morrison, 19 85, Science 229:1202-7; Oi and Morrison, 1986, BioTechniques 4:214-221; Gillies et al., 1989, J Immunol. Methods 125:191-202; and U.S. 5,807,715 Specification No. 4,816,567, Specification No. 4,816,397 and the same (See Specification No. 6,331,415).

[0155] Methods for producing humanized antibodies are well known in the art (e.g., international publications). Pamphlet No. 91 / 09967; Padlan, 1991, Mol Immunol 28(4 / 5): 489-498; St udnicka et al, 1994, Prot Engineering 7(6): 805-814; Roguska et al, 1994, PNAS 9 1: 969-973; International Publication No. 93 / 17105 pamphlet; Tan et al, 2002, J Immunol 169: 1119-25; Caldas et al, 2000, Protein Eng. 13(5): 353-60; Morea et al, 2000, Methods 20(3): 267-79; Baca et al, 1997, J Biol Chem 272(16):10678-84; Roguska et al, 1996, Protein Eng 9(10): 895 904; Couto et al, 1995, Cancer Res. 55 (23 S upp): 5973s-5977s; Couto et al, 1995, Cancer Res 55(8): 1717-22; Sandhu, 1994, G See ene 150(2):409-10; Pedersen et al, 1994, J Mol Biol 235(3): 959-73. For example, Humanized antibodies can be produced by CDR grafting.

[0156] Methods for producing human antibodies are well known in the art. For example, human antibodies are produced by... As described above, using an antibody library derived from human immunoglobulin sequences, phages It can be produced by display or yeast surface display method. (United States) Specification No. 4,444,887, Specification No. 4,716,111 and Specification No. 5,885, Specification No. 793; International Publication No. 98 / 46645 pamphlet, International Publication No. 98 Pamphlet No. 50433, International Public Gazette No. 98 / 24893, International Public Gazette Publication No. 98 / 16654, International Publication No. 96 / 34096 International Publication No. 96 / 33735, and International Publication No. 91 / 1 See also pamphlet No. 0741. Furthermore, human antibodies are used in mouse-human hybridomas. It can also be manufactured using (Shinmoto et al, 2004, Cytotechnology 46: 19-23; See Naganawa et al, 2005, Human Antibodies 14: 27-31.

[0157] Methods for producing antibody fragments are well known in the art. For example, Fab fragments and F( The ab')2 fragment is produced by pepsin (to generate the F(ab')2 fragment) or papain ( Protein degradation of immunoglobulin molecules using enzymes such as those used to generate Fab fragments. It can be generated by cleavage. Methods for producing scFv fragments are also known in the art. (For example, Ahmad et al., 2012, Clinical and Developmental Immunology, doi: 10.1155 / 2012 / 980250; Wang et al., 2006, Anal. Chem. 78, 997-1004; Pansri et al., 2009, BMC Biotechnology 9:6; Chao et al., 2006, Nature Protocols 1(2):755-768 (See reference). scFv with desired antigen-binding properties can be displayed using phage display technology or yeast. It can be selected by surface display technology. scFv is a short polypeptide chain. The process of fusing the variable domains of the heavy and light chains of immunoglobulins via a linker (recombination). It can be constructed using expression methods. Single-domain antibodies (e.g., light Methods for producing antibodies lacking a chain are well known in the art (e.g., Riechmann & Muyl dermans, 1999, J Immunol 231:25- 38; Nuttall et al, 2000, Curr Pharm Biotechnol See 1(3):253-263; Muyldermans, 2001, J Biotechnol 74(4): 277-302).

[0158] Methods for producing bispecific antibodies are well known in the art (for example, Konterma See n, 2012, MAbs 4: 182-197; Gramer et al., 2013, MAbs 5: 962-973).

[0159] If the selected CDR amino acid sequence is a short sequence (e.g., less than 10-15 amino acids in length) In one embodiment, the nucleic acid encoding the CDR is, for example, Shiraishi et al. (2007) Nuclei c Acids Symposium Series 51(1):129-130 and U.S. Patent No. 6,995,259 It can be chemically synthesized as described in [reference]. A given nucleus encoding an acceptor antibody Regarding the acid sequence, the region of the nucleic acid sequence encoding the CDR was analyzed using standard molecular biology techniques. It can be replaced with chemically synthesized nucleic acids using [method]. The 5' and [another part of the chemically synthesized nucleic acid] The 3' end is used when cloning that nucleic acid into the nucleic acid that encodes the variable region of the donor antibody. It can be synthesized to include a sticky-end restriction enzyme site for use in [the application of the enzyme].

[0160] In some embodiments, the antibody described herein is the corresponding unmodified antibody. It has an effector function that is enhanced or reduced compared to the normal range (or an effector unit) The constant region of the antibody described herein is: The effects functions involved are adjusted by changing the characteristics of the steady-state or Fc region. It can be modified. Examples of modified effector functions include the following activity: antibody Complement-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), apoptosis, monophasic It binds to multiple Fc receptors and modulates one or more of the pro-inflammatory responses. The section is given. The regulation is modified compared to the activity of the constant region in the unmodified form. The increase or decrease in effector functional activity shown by the target antibody containing the constant region, This refers to the disappearance of FcR binding affinity and / or ADCC activity and / or The modified constant region has altered CDC activity, while the constant region is in an unaltered form. Compared to the region, FcR binding activity and / or ADCC activity are enhanced or reduced. / A polypeptide that has either CDC activity or shows increased binding to FcR. The modified constant region has at least one FcR compared to the unmodified polypeptide. It also binds with high affinity. The modified constant region, which shows a decrease in binding to FcR, has been modified. It binds to at least one FcR with lower affinity than the constant region in the non-constant form.

[0161] Methods for conferring CDC activity or ADCC activity to antibodies are well known in the art (e.g.) For example, Kellner et al., 2014, Methods 65: 105-113; International Publication No. 2012010562. Pamphlet; Natsume et al., 2009, Drug Design, Development and Therapy 3(3):7- See 16). Such methods include Fc isotype shuffling and enhanced CDC. and / or amino acid mutations in the Fc region that confer ADCC activity, as well as enhancement The Fc region glycosylation profile that confers CDC and / or ADCC activity This includes, but is not limited to, changes.

[0162] For example, the antibodies described herein enhance or reduce complement-dependent cell cytotoxicity (C It may contain a modified constant region that exhibits DC activity. The regulated CDC activity is the F of the antibody. This is achieved by introducing one or more amino acid substitutions, insertions, or deletions in the c region. It is possible. See, for example, U.S. Patent No. 6,194,551. Alternatively, Additionally, a cysteine ​​residue is introduced into the Fc region, thereby creating an interchain relationship in this region. It can also enable the formation of disulfide bonds. The homodimer thus produced Somatic antibodies have improved or reduced internalization capabilities, and / or Complement-mediated cell killing may be increased or decreased. For example, Caron et al. (19 92) J Exp Med 176:1191-1195 and Shopes (1992) Immunol 148:2918-2922; PCT published. International Publication No. 99 / 51642 and International Publication No. 94 / 29351 Lett; Duncan and Winter (1988) Nature 322:738-40; and U.S. 5,648, See Specification No. 260 and Specification Nos. 5,624,821.

[0163] Any of the antibodies described herein may be used in any immunology or biochemistry known in the art. Using scientifically based methods, antigens can be detected either in vitro or in vivo. For example, NPM1c: modulates either activity or function caused by HLA-A2. The ability can be screened and / or tested.

[0164] The antibodies or antigen-binding fragments described herein are used in molecular biology and proteinogenesis. It can be prepared using various methods known in the field of science and technology. For example, antibody heavy chains And nucleic acids encoding one or both of the light chain polypeptides, for example, in a promoter Column, ribosome binding site, transcription start sequence and transcription stop sequence, translation start sequence and translation stop sequence Stop sequences, transcription terminator signals, polyadenylation signals, and enhancers Expression containing transcriptional regulatory sequences and translational regulatory sequences, including sequences or activator sequences. It can be inserted into a vector. The regulatory sequence includes a promoter and a transcription initiation sequence. and transcription stop sequences are included. In addition, the expression vector is such that it can be expressed in two different organisms. For example, in mammalian or insect cells for expression, as well as clonin It may include multiple replication systems so that they can be maintained in a prokaryotic host for replication and amplification. ru.

[0165] Cloned heavy and light chain polypeptides from nucleic acids in mammalian cells Several possible vector systems can be used for expression. One class relies on the integration of the desired gene sequence into the host cell genome. Cells containing the implanted DNA have drug resistance genes, for example, E. coli g pt (Mulligan and Berg (1981) Proc Natl Acad Sci USA 78:2072) or Tn5 n By simultaneously introducing eo (Southern and Berg (1982) Mol Appl Genet 1:327) You can choose to select a marker gene. The selection marker gene is a DNA gene that you want to express. They may be linked in a row, or introduced into the same cells by cotransfection. This is also possible (Wigler et al. (1979) Cell 16:77). The second class of vectors is extrachromosomal. These utilize DNA elements that confer self-renewal ability to plasmids. These vectors are Animal viruses, for example, bovine papillomavirus (Sarver et al. (1982) Proc Natl Aca d Sci USA, 79:7147), cytomegalovirus, polyomavirus (Deans et al. (19 84) Proc Natl Acad Sci USA 81:1292), or SV40 virus (Lusky and Botchan This may originate from (1981) Nature 293:79).

[0166] Expression vectors can be introduced into cells in a manner suitable for the subsequent expression of nucleic acids. The method of introduction is mainly determined by the type of target cell, as described below. Typical methods include CaPO4 precipitation, liposome fusion, cationic liposomes, and electrolysis. Electroporation, viral infection, dextran-mediated transfection, polybren-mediated transfection Transfection, protoplast fusion, and direct microinjection are included. Born.

[0167] Suitable host cells for the expression of antibodies or their antigen-binding fragments include yeast, bacteria, insects, This includes plant and mammalian cells. Of particular interest are *E. coli* cells. i) Bacteria such as Saccharomyces cerevisiae True Pichia visiae and Pichia pastoris Fungi, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), and primary cells. It is a stock.

[0168] In some embodiments, the antibody or a fragment thereof is used in transgenic animals (e.g., tigers). It can be expressed in mutant mammals and purified from there. For example, Antibodies were prepared by Houdebine (2002) Curr Opin Biotechnol 13(6):625-629; t al. (2000) Transgenic Res 9(2):155-159; and Pollock et al. (1999) J Immunol M As described in ethods 231(1-2):147-157, transgenic non-human mammals It can be produced in (for example, rodents) and isolated from milk, for example.

[0169] Antibodies and their fragments are expressed using an expression vector containing nucleic acids encoding the antibody or fragment. The transformed host cells are then subjected to conditions sufficient to enable protein expression. Proteins can be produced from cells by culturing them for a certain length of time. Such conditions for protein expression vary depending on the expression vector and host cell selection. This is thought to be the case and would be easily confirmed by those skilled in the art through conventional experiments. For example, Refolding antibodies expressed in Escherichia coli (E. coli) from inclusion bodies. This is possible (see Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and their Methods for use are well known in the art (Current Protocols in Molecular Biolo gy, Wiley & Sons, and Molecular Cloning--A Laboratory Manual --3rd Ed., Cold Spr. See ing Harbor Laboratory Press, New York (2001). Codon, suitable expression vector. And the selection of suitable host cells varies depending on several factors, and can be easily adjusted as needed. It can be optimized. The antibodies (or fragments thereof) described herein can be used in mammalian cells. , or including but not limited to yeast, baculovirus, and in vitro expression systems. It is not expressed in other expression systems (for example, Kaszubska et al. (20 00) See Protein Expression and Purification 18:213-220).

[0170] After expression, the antibody and its fragments can be isolated. The antibody or its fragments can be tested. Depending on what other components are present in the material, isolation can be performed using various methods known to those skilled in the art. Alternatively, it can be purified. Standard purification methods include electrophoretic, molecular, and immunological methods. Methods, as well as ion exchange, hydrophobicity, affinity, and reverse-phase HPLC chromatography. This includes chromatography techniques, including fees. For example, antibodies are standard anti-antibody assays. Purification can be performed using a lamb (for example, a Protein A or Protein G column). Ultrafiltration and diafiltration techniques, combined with protein concentration, are also used. It is useful. For example, Scopes (1994) “Protein Purification, 3rd edition,” Springer -See Verlag, New York City, New York. The required degree of purification varies depending on the desired use. It is thought that in some cases, purification of the expressed antibody or its fragments is not necessary. .

[0171] Methods for determining the yield or purity of purified antibodies or fragments thereof are available in the art. This is publicly known, for example, by the Bradford assay, UV spectroscopy, and Biuret protein. Assays, Lowry protein assays, Amido Black protein assays, high-pressure solutions Body chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis (examples) For example, protein staining, such as Coomassie blue or colloidal silver staining, It is included.

[0172] Antibodies or their antigen-binding fragments can be modified after their expression and purification. The modification can be covalent or noncovalent. Such modifications are, for example, The target amino acid residue of the lipeptide can be reacted with a selected side chain or terminal residue. It can be introduced into antibodies or fragments by reacting it with an organic derivatizing agent. Suitable sites for modification include, for example, structural analysis or amino acid sequence analysis of the antibody or fragment. You can select using any of the following criteria, including:

[0173] In some embodiments, the antibody or its antigen-binding fragment is conjugated to a heterologous portion. This is possible. The heterogeneous portion can be, for example, a heterogeneous polypeptide, a therapeutic agent, or a cytotoxic agent. Agents (e.g., toxins or drugs), or detectable labels, e.g., but not limited to, Injection labeling, enzyme labeling, fluorescent labeling, heavy metal labeling, luminescence labeling, or biotin or strene It could be an affinity tag such as ptavidin. Suitable heterologous polypeptides include, for example, For example, antigen tags (e.g., FLAG(DYKDD)) for use in the purification of antibodies or fragments. DDK (SEQ ID NO: 44), polyhistidine (6-His; HHHHHH (SEQ ID NO: 45) ), hemagglutinin (HA;YPYDVPDYA (SEQ ID NO: 46)), glutathione-S - Contains transferase (GST) or maltose-binding protein (MBP) Heterogeneous polypeptides are also useful as diagnostic or detection markers. Peptides (e.g., enzymes), luciferase, fluorescent proteins (e.g., green fireflies) Photoprotein (GFP), or chloramphenicol acetyltransferase ( This also includes CAT. Suitable radioactive labels include, for example, 32P, 33P, 14C, and 125. It includes I, 131I, 35S, and 3H. Suitable fluorescent labels include fluorescein. Fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), Dy Light (trademark) 488, phycoerythrin (PE), propidium iodide (PI), PerCP, PE-Alexa Fluor(registered trademark) 700, Cy5, Aloficococcus This includes, but is not limited to, anine and Cy7. Examples of bioluminescent markers include, for example, species Contains one of the following luminescent lanthanides (e.g., europium or terbium) chelates For example, suitable europium chelates include diethylenetriaminepentaacetic acid (DT PA) or tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) It contains hopium chelate. Enzyme labeling can be done using, for example, alkaline phosphatase, CAT. It contains luciferase and horseradish peroxidase.

[0174] Two proteins (e.g., an antibody and a heterologous portion) crosslink with several known chemical crosslinking agents. Crosslinking can be done using either of these. An example of such a crosslinking agent is "interfered" This involves linking two amino acid residues via a linkage that includes a sulfide bond. In linkage, the disulfide bond within the crosslinking unit is, for example, reduced glutathione or yeast From reduction by the action of disulfide reductase, (due to interfering groups on both sides of the disulfide bond) (and) protected. One suitable reagent is 4-succinimidyloxycarbonyl-α -Methyl-α(2-pyridyldithio)toluene (SMPT) is at the end of one protein. By utilizing the terminal cysteine ​​of lysine and the other, such They form links. Heterobinary proteins that cross-link through different coupling sites on each protein. Potential crosslinking agents can also be used. Other useful crosslinking agents include those that link two amino groups. Drugs (for example, N-5-azido-2-nitrobenzoyloxysuccinimide), two s Reagents for linking rhudyl groups (e.g., 1,4-bis-maleimidobutane), amino groups A reagent that links the sulfhydryl group (for example, m-maleimidebenzoyl-N-hydro (Xysuccinimide ester), reagents that link amino groups and carboxyl groups (for example, 4-[p-azidosalicylamide]butylamine), and the amino group and arginine side chain A reagent that links with the guanidinium group present (for example, p-azidophenylglyoxa This includes, but is not limited to, ethanol monohydrate.

[0175] In some embodiments, the radiolabel is directly conjugated onto the amino acid backbone of the antibody. Alternatively, a radiolabel can be attached to a free amino group to meta-label the associated protein. Larger molecules that form iodophenyl (mIP) derivatives (e.g., meta-[125I ] Iodophenyl-N-hydroxysuccinimide ([125I]mIPNHS) 5I) (See, for example, Rogers et al. (1997) J Nucl Med 38:1221-1229) or Kirei It can be included as part of a drug (e.g., DOTA or DTPA), and then it can be used as a protein. It may be bonded to the skeleton. Radiolabeled or larger molecules / chelating agents containing it. A method for conjugating an antibody or antigen-binding fragment as described herein is the technique This is well known in the field. Such methods involve radiolabeling or chelating proteins. Under conditions that promote binding (e.g., pH, salt concentration, and / or temperature), the protein This involves radiolabeling and incubation (e.g., U.S. Patent No. 6,001,329). (See the specifications.)

[0176] Fluorescent labels (sometimes called "fluorophores") are used to label proteins (e.g., antibodies). Methods for conjugating ) are well known in the field of protein chemistry. For example, f The ruolophore is bound to the succinimidyl (NHS) ester or It uses the tetrafluorophenyl (TFP) ester moiety to release the free amino acids of the protein. A conjugate to a group (e.g., lysine) or a sulfhydryl group (e.g., cysteine). It can be made to work. In some embodiments, the fluorophore is sulfo-SMCC Which heterobifunctional crosslinking agent portion can be conjugated? Suitable conjugate The fluorophores binding method involves binding antibody proteins, or fragments thereof, to proteins. This involves incubation with fluorophores under conditions that promote the synthesis. For example, Welch and Redvanly (2003) “Handbook of Radiopharmaceuticals: Radiochemistry and Appli See "cations," John Wiley and Sons (ISBN 0471495603).

[0177] In some embodiments, antibodies or fragments are, for example, in circulation, such as in blood, serum, and This involves modifying the material with a portion that improves the stabilization and / or retention of antibodies in other tissues. This can be done. For example, an antibody or fragment can be used, for example, Lee et al. (1999) Bioconjug Chem 10( 6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; As described in Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 It can be converted to PEG or HES as shown (Fresenius Kabi, Germ any; see, for example, Pavisic et al. (2010) Int J Pharm 387(1-2):110-119). Stabilization section This improves the stability or retention of the antibody (or fragment) by at least approximately 1.5 times (for example, less At least 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, or 50 times It can be improved by two or more times.

[0178] In some embodiments, the antibody or antigen-binding fragment described herein is glycosylated It can be converted into an antibody or antigen as described herein. In some embodiments, the antibody or antigen described herein can be converted into an antigen. The binding fragment is reduced or absent in the glycosylation of the antibody or the fragment. It can be subjected to enzymatic or chemical processing, or produced from cells. Methods for producing antibodies with reduced cosylation are known in the art, for example, National Patent No. 6,933,368; Wright et al. (1991) EMBO J 10(10):2717-2723 This is described in and Co et al. (1993) Mol Immunol 30:1361.

[0179] bispecific molecules In certain embodiments, antigen-binding constructs that can be used to form bispecific molecules may be used. The following substances are provided herein: MHC proteins (e.g., anti-NPM1c:MHC Mutant nucleophosmin protein neoepitope that forms a complex with a Class I antibody. An antibody against an antigen containing the antigen, or its antigen-binding fragment, is derivatized or otherwise modified by another molecule. For example, another peptide or protein (e.g., a ligand for another antibody or receptor) ) is linked to and binds to at least two different binding sites or target molecules, resulting in bispecificity. Molecules can be generated. For example, anti-NPM1c:HLA-A2 antibody, or its anti- The primordial binding fragment (e.g., scFv) is placed on T cells (e.g., CD3) or natural killer cells. -An antibody that specifically binds to an antigen expressed on a cell, or an antigen-binding fragment thereof (e.g., It can be linked to scFv). More than two different binding sites and / or targets. Derivatization of a multispecific molecule that binds to a molecule, or as described herein It is produced by linking an antibody, or its antigen-binding fragment, to multiple other molecules. It is also possible; such multispecific molecules are also referred to as "bispecific molecules" as used herein. It is intended to be included in the scope of the term. The bispecific molecules described herein To produce it, two antibodies, or antigen-binding fragments thereof, as described herein, To create a hemispecific molecule, one or more other molecules, such as another antibody or antibody fragment, are required. It is preferable to link them to peptides or binding mimetic bodies (for example, chemical coupling or co (Through densification, gene fusion, non-covalent association, etc.)

[0180] Therefore, in certain embodiments, MHC proteins (e.g., NPM1c:HL) are used. Contains a mutant nucleophosmin protein neoepitope that forms a complex with A-A2. At least one first binding specificity to the antigen (i.e., the first of the bispecific molecules) (Antigen-binding domain) and a second binding specificity to a second target epitope (i.e., A bispecific molecule containing the second antigen-binding domain of a bispecific molecule is described herein. Provided. In embodiments in which the bispecific molecules described herein are multispecific, The offspring may further include a third binding specificity.

[0181] In a particular embodiment, the first antigen-binding domain of the bispecific molecule and the bispecific molecule The specificity of the second antigen-binding domain is the same. In certain embodiments, bispecificity is observed. The specificity of the first antigen-binding domain of the molecule and the second antigen-binding domain of the bispecific molecule are different. Yes.

[0182] In one embodiment, the bispecific molecule described herein is at least one antibody The body, or a fragment of its antibody, e.g., Fab fragment, Fab' fragment, F(ab')2 fragment, F Examples include v fragments, single-chain Fv (scFv), or single-chain antibody molecules. The content of Ladner et al. is incorporated herein by reference throughout. As described in U.S. Patent No. 4,946,778, antibodies are light chain dimers. Alternatively, a heavy chain dimer, or any very small fragment thereof, such as Fv or single chain construction. It can be an object.

[0183] In certain embodiments, the bispecificity molecule of this disclosure is a bispecificity single-chain antibody. In the embodiments, at least one of the antigen-binding domains in the bispecificity molecule of the Disclosure One is a single-chain fragment of the variable region of an antibody.

[0184] In certain embodiments, the antibody used in the bispecific molecule described herein The antigen-binding fragment is human (e.g., a human monoclonal antibody). Other antibodies that may be used in the listed bispecific molecules include mouse antibodies, chimeric antibodies, or human antibodies. These are monoclonal antibodies (e.g., mouse, chimeric, or humanized monoclonal antibodies).

[0185] The bispecific molecules described herein have an antigen-binding domain as a component of the technology It can be prepared by conjugating using methods known in the field. Example For example, by generating each antigen-binding domain of a bispecific molecule separately, and then conjugating them together... It can be gated.

[0186] When the antigen-binding domain is a protein or peptide, various coupling agents and Crosslinking agents can be used for covalent conjugation. Examples of crosslinking agents These include protein A, carbodiimide, and N-succinimidyl-S-acetylthio. Acetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-Phenylenedimaleimide (oPDM), N-Succinimidyl-3-(2-Pyridyl) Dithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maley Examples include midomethyl)cyclohexane-1-carboxylic acid ester (sulfo-SMCC). (e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) See Proc. Natl. Acad. Sci. USA 82:8648. For other methods, see Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Gle This includes what is described in nnie et al. (1987) J. Immunol. 139: 2367-2375. The most suitable conjugation agents are SATA and sulfo-SMCC, both of which are Pi It is available from Erce Chemical Co. (Rockford, IL).

[0187] When antigen-binding specificity is an antibody, they are sulfurous in the C-terminal hinge region of the two heavy chains. They can be conjugated via drill coupling. In a particularly preferred embodiment, The radiating region has an odd number, preferably one, sulfhydril residues prior to conjugation. It is modified to include the group.

[0188] Alternatively, both antigen-binding domains can be encoded by the same vector, and the same host It may also be expressed and assembled within cells. In this method, the bispecific molecules are mAb and m Ab, mAb and Fab, mAb and Fab', mAb and F(ab')2, mA b and Fv, mAb and scFv, Fab and F(ab')2, Fab and Fa b, Fab' and Fab', F(ab')2 and F(ab')2, scFv and s When including cFv, Fv and Fv, or ligand and Fab fusion protein, This is useful for [purpose]. Bispecific antibodies may also include antibodies containing scFv at the C-terminus of each heavy chain. The bispecific molecules described herein contain one single-chain antibody and one binding determinant. It may be a single-chain molecule, or a single-chain bispecific molecule containing two binding determinants. The antibody may include antibodies containing scFv at the N-terminus of each heavy chain. Bispecific antibodies include each light The antibody may contain scFv at the N-terminus or C-terminus of the chain. Bispecific molecules are few. It may contain at least two single-chain molecules. A method for preparing a bispecific molecule is, for example, U.S. Patent No. 5,260,203; U.S. Patent No. 5,455,030; U.S. Japanese Patent No. 4,881,175; U.S. Patent No. 5,132,405; U.S. Patent Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. U.S. Patent No. 5,013,653; U.S. Patent No. 5,258,498; and U.S. Patent It is described in Specification No. 5,482,858.

[0189] The binding of a bispecific molecule to its specific target is done by methods recognized in the art, for example, Enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS Using analysis, bioassays (e.g., growth inhibition), or Western blot assays This can be confirmed by doing so. Each of these assays generally targets the complex of interest. By using a specific labeling reagent (e.g., an antibody), the target protein-antibody can be identified. The presence of the body complex is detected.

[0190] In some embodiments, the bispecific molecule of this disclosure is an MHC class I protein (e.g., Mutant nucleophosmin protein neoepitot (HLA-A2) that forms a complex with HLA-A2. It binds to antigens containing (e.g., specifically), and at the same time, immune effector cells, e.g. For example, T cells (e.g., CD3) or natural killer cells (e.g., NKp46) It also binds to one or more antigens on CD16A (e.g., specifically). In some embodiments, the bispecific molecules of this disclosure are specific to NPM1c:HLA-A2. It binds and, at the same time, specifically binds to one or more antigens on immune effector cells. In some embodiments, such binding leads to the retargeting of immune effector cells into tumor cells. To enable (see, for example, Chames et al., 2009, MAbs 1:539-547). Immuno-effectors -Cells include T cells, natural killer cells, macrophages, neutrophils, and dendritic cells. Examples include, but are not limited to, B lymphocytes. In some embodiments, as described herein The immune effector cells targeted by the bispecific antibodies are T cells (for example). , CD3), natural killer cells (e.g., NKp46 or CD16A), or m It is a clophage. In some embodiments, the bispecific molecule is NPM1c:HLA-A 2. An antibody or its antigen-binding fragment (e.g., scFv) that binds to 2, and an immunoeffector. - Cells, for example, T cells (e.g., CD3), natural killer cells (e.g., NKp4) Antibodies that bind to antigens on macrophages (6 or CD16A), or antibodies that bind to antigens on macrophages or their antigenic binding Includes combined fragments (e.g., scFv).

[0191] In some embodiments, the bispecific molecule of this disclosure is an MHC class I protein (e.g., Mutant nucleophosmin protein neoepitot (HLA-A2) that forms a complex with HLA-A2. It binds to antigens containing (e.g., NPM1c neoepitope) (e.g., it binds specifically) ), and simultaneously, the following antigens: CD3, NKp46, CD16A, CD40, CD47, 4- 1BB, TGF-β, LAG-3, PD-1, TIM-3, CTLA-4, OX-40, It may also bind to one or more of NKp30, NKG2A, NKG2D, or DNAM-1. To bind (for example, to bind specifically). In one embodiment, the bispecific molecule of the present disclosure is C It includes a second binding specificity to D3. In one embodiment, the bispecificity molecule of the present disclosure is N It includes a second binding specificity to Kp46. In one embodiment, the bispecificity molecule of the present disclosure is , including a second binding specificity to CD16A. In one embodiment, the dual specificity component of the present disclosure The child includes a second binding specificity to CD40. In one embodiment, the dual specificity of the present disclosure The molecule contains a second binding specificity to CD47. In one embodiment, the dual specificity of the disclosure The sex molecule contains a second binding specificity to 4-1BB. In one embodiment, the dual of the present disclosure The specificity molecule includes a second binding specificity to TGF-β. In one embodiment, the present disclosure The bispecific molecule contains a second binding specificity to LAG-3. In one embodiment, The shown bispecific molecule contains a second binding specificity to PD-1. In one embodiment, The disclosed bispecific molecule includes a second binding specificity to TIM-3. In one embodiment, The bispecific molecule of this disclosure contains a second binding specificity to CTLA-4. In this state, the bispecific molecule of this disclosure includes a second binding specificity to OX-40. In its application form, the bispecific molecule of this disclosure includes a second binding specificity to NKp30. In one embodiment, the bispecific molecule of this disclosure contains a second binding specificity to NKG2A. In one embodiment, the bispecific molecule of the present disclosure has a second binding specificity to NKG2D. Includes. In one embodiment, the bispecific molecule of the present disclosure has a second binding to DNAM-1. Includes specificity.

[0192] In some embodiments, the bispecific molecule may be a bispecific single-chain antibody. The terms "specific single-chain antibody" or "single-chain bispecific antibody" refer to antibodies that are completely identical to immunoglobulins. In a single polypeptide chain, at least one constant moiety and / or Fc moiety is lacking. This refers to an antibody construct resulting from connecting two antibody variable regions. For example, a bispecific mono-antibody. Each antigen-specific portion of the chain antibody is antibody V H Region and antibody V L Includes the region.

[0193] The advantageous variants of the bispecific single-chain antibody are listed below from the N-terminus to the C-terminus (" "CD3" is used as a second example of specificity, but other antigens, such as NKp46, CD16A, CD40, CD47, 4-1BB, TGF-β, LAG-3, PD-1, T IM-3, CTLA-4, OX-40, NKp30, NKG2A, NKG2D, or DN (To be replaced by AM-1) V L (NPM1c:HLA-A2)-V H (NPM1c:HLA-A2)-V H (CD 3)-V L (CD3) V H (NPM1c:HLA-A2)-V L (NPM1c:HLA-A2)-V H (CD 3)-V L (CD3) V L (NPM1c:HLA-A2)-V H (NPM1c:HLA-A2)-V L (CD 3)-V H (CD3) V H (NPM1c:HLA-A2)-V L (NPM1c:HLA-A2)-V L (CD 3)-V H (CD3) V H (CD3)-V L (CD3)-V H (NPM1c:HLA-A2)-V L (NPM 1c: HLA-A2), V H (CD3)-V L (CD3)-V L (NPM1c:HLA-A2)-V H (NPM 1c: HLA-A2), V L(CD3)-V H (CD3)-V H (NPM1c:HLA-A2)-V L (NPM 1c:HLA-A2), or V L (CD3)-V H (CD3)-V L (NPM1c:HLA-A2)-V H (NPM 1c:HLA-A2).

[0194] The antigen-binding domains of the bispecific molecules of the present disclosure preferably have a specificity that is at least substantially identical to, for example, the binding specificities of the antibodies or immunoglobulin chains from which they are derived and do so

[0195] In certain embodiments, the antigen-binding domains of the bispecific molecules that bind to one or more antigens on immune effector cells, such as T cells (e.g., CD3) or natural killer cells (e.g., NKp46 or CD16A), have a binding affinity (Kd) for the antigen of at least 1 0 0 -4 M, at least 10 -5 M, at least 10 -6 M, or at least 10 -7 M In certain embodiments, the antigen-binding domains that bind to one or more antigens on immune effector cells, such as , T cells (e.g., CD3) or natural killer cells (e.g., NKp46 or C D16A), have a binding affinity (Kd) for the antigen that is not stronger than 1 0 -7 M (e.g., between 10 -4 M and 10 -7 M, or between 10 -5 M and 1 0 -7 M). In certain embodiments, NPM1c: The antigen-binding domain of the bispecific molecule that binds to HLA-A2 antigen is NPM1c:HLA -A2 antigen, with at least 10 -7 M or stronger, at least 10 -8 M or even stronger, at least 10 -9 M or stronger, at least 500 nM or stronger, at least 250 nM or stronger, at least 100 nM or stronger, at least 50 nM or stronger, at least 25 nM or stronger, at least 20 nM or stronger, at least 15 nM or stronger, or at least 10 nM or stronger, having a binding affinity (Kd) of at least 15 nM or stronger, or at least 10 nM or stronger. In certain embodiments, the antigen-binding domain of the bispecific molecule that binds to NPM1c:HLA-A 2 antigen has a binding affinity (Kd) of at least 20 nM or stronger, at least 15 nM or stronger, or at least 10 nM or stronger for the NPM1c:HLA-A2 antigen. In certain specific embodiments , the antigen-binding domain of the bispecific molecule that binds to the NPM1c:HLA-A2 antigen is between 0.1 nM and 500 nM, 0.1 nM and 10 0 nM, 0.5 nM and 100 nM, 0.1 nM and 50 nM, 0.5 nM and 50 nM, 0. 1 nM and 25 nM, 0.5 nM and 25 nM, 0.1 nM and 15 nM, 0.5 nM and 15 nM , 0.1 nM and 10 nM, or 0.5 nM and 10 nM (or from the previous value to the subsequent value), or has a binding affinity (Kd ) between 1 nM and 100 nM (or any value therebetween) for the NPM1c:HLA-A2 antigen. Such antigen-binding domains have, for an antigen on immune effector cells (e.g., CD3 antigen), at least 10 M, e.g., not stronger than 10 -5 M -7 or weaker binding It can have affinity for NPM1c:HLA-A2 antigen, for example, 10 - 8 M or stronger, 50nM or stronger, 25nM or stronger, 15nM or It can have a stronger binding affinity of 10 nM or more. The dual feature of this disclosure In certain embodiments of the isomer molecule, (a) the binding site (N) of the first antigen-binding domain PM1c (which binds to MHC class I) is at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 500 nM, at least about 100 nM, At least approximately 50 nM, or at least approximately 25 nM, or at least approximately 15 nM (b) has affinity for; and / or (b) the binding site of the second antigen-binding domain is approximately 1 0 -7 Weaker than M, about 10 -6 Weaker than M, or 10 -5 Affinity of the M order In some embodiments of the bispecific molecules of this disclosure, (a) a first antigen-binding domain The aforementioned binding site (NPM1c: binds to HLA-A2) has a molecular weight of at least approximately 100 nM. or having an affinity of at least about 25 nM; and / or (b) a second antigen-binding agent The main connection point is approximately 10 -7 Weaker than M, about 10 -6 Weaker than M, or 1 0 -5 It has affinity of the order M.

[0196] According to a particular embodiment mentioned above, MHC proteins (e.g., NPM1c) Antigens containing mutant NPM1c neoepitopes that form complexes with HLA-A2 antigens For the recognition binding site to capture the target cell it is trying to destroy with high efficiency, high parental Having compatibility is advantageous. On the other hand, antigens on immune effector cells (e.g., CD3) The binding affinity of the binding site that recognizes the antigen is the same as that of the innate receptor for the antigen (e.g., CD3 receptor). The binding affinity of the receptor (or immune effector cell receptor, e.g., T cell receptor) Regarding the interaction with ligands, i.e., MHC-peptide complexes on the surface of target cells, This could be of an order of commonly observed binding affinity.

[0197] In one embodiment of the present disclosure, the first and / or second of the bispecific molecules of the present disclosure The main component is V derived from natural antibodies. H Region and V L To mimic the domain or to correspond to it The antibody that provides a binding site for the bispecific molecule of this disclosure is, for example, monoclonal. Antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, synthetic antibodies, antibodies Fragments, such as Fab, Fv, or scFv fragments, or any of these chemicals It may be a modified derivative. Monoclonal antibodies are, for example, Kohler and Milstei n, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3 were the first to publish this information. It can be prepared by the described method, which is mouse myeloma cells, in the art. Therefore, by fusing it with spleen cells derived from mammals that have undergone immunization with the developed modification, This includes, for example, antibodies or fragments of the aforementioned antigens, such as those used by Harlow and Lan. e "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988 Antibodies can also be obtained by using established methods. Antibodies include several types, including human. It can be obtained from seeds. Derivatives of the antibody can be obtained by phage display. In such cases, surface plasmon resonance used in the BIAcore system is used to NP M1c: It can increase the efficiency of phage antibodies that bind to HLA-A2 or a secondary antigen. (Schier, Human Antibodies Hybridomas 7 (1996), 97 105; Malmborg, J. Immunol Methods 183 (1995), 7 13). For example, the production of chimeric antibodies is described in International Publication No. 89 / 096. It is described in pamphlet No. 22. Methods for producing humanized antibodies are, for example, in Europe. Patent Application Publication No. 10239400 and International Publication No. 90 / 07861 Further sources of the antibodies used in accordance with this disclosure are listed in the brochure. These are heterologous antibodies. General principles regarding the production of heterologous antibodies, for example, human antibodies in mice. For example, see International Publication No. 91 / 10741, International Publication No. 94 / 0260 Pamphlet No. 2, International Publication No. 96 / 34096 Pamphlet and International Publication No. 96 / As described in Brochure No. 33735. Another supply of antibodies used in accordance with this disclosure. The source is, for example, Chao et al., 2006, Nature Protocols 1(2):755-768. These are human antibodies isolated and manipulated using sea urchin and yeast surface display.

[0198] In one embodiment, the bispecific molecule described herein has the NPM1c:HLA-A2 characteristic The heterogeneous domain is represented as sequence number 11 (ARLGYPTTTLLPFDY). V contains or is the noacid sequence H CDR3, Sequence ID 10 ( The amino acid sequence is represented as ISGSGGST, or contains at least that amino acid sequence. One V H As shown in CDR2 and / or Sequence ID 9 (GFTFSSYA) At least one V containing or having an amino acid sequence H Includes CDR1.

[0199] The bispecific molecules of this disclosure have one or more NPM1c:HLA-A2 specific domains. The number V L Regional CDR may also be included. NPM1c:HLA-A2 specific domain UnaV L The region CDR is the amino acid sequence shown as Sequence ID No. 8 (QQSYSTPLT). at least one V that contains or is L CDR3, Sequence ID 7 (A AS) At least one V containing or having an amino acid sequence L CDR2, and / or or contains the amino acid sequence shown as sequence number 6 (QSISSY) or at least one V L Includes CDR1.

[0200] In one embodiment, the bispecific molecule is a single construct (e.g., C described herein). In DR, the antibody V specifically binds to NPM1c:HLA-A2. H and / or V L Includes CDR1, CDR2, and CDR3.

[0201] In one embodiment, the anti-NPM1c:HLA-A2 CDR referred to herein is IM It follows the GT numbering system. The IMGT numbering scheme is based on the consistency of residues in antibodies. This is a widely adopted standard for numbering in this format (IMGT® registered trademark, i international ImMunoGeneTics information system(registered trademark) website imgt.org, founder and director: Marie-Paul See Lefranc, Montpellier, France; for example, Lefranc, M.-P., 1999, The Immunolo gist, 7: 132-136 and Lefranc, M.-P. et al., 1999, Nucleic Acids Res., 27:209-21 See section 2. Both of these are incorporated herein by reference in their entirety.

[0202] In certain embodiments, the bispecific molecules of this disclosure (e.g., bispecific antibodies or cleavage) One of the molecules binds simultaneously to NPM1c:HLA-A2 and CD3. In some embodiments, The bispecific molecule is a single-chain variable (scFv) fragment that binds to NPM1c:HLA-A2. and includes an antibody that binds to CD3 or an antigen-binding fragment thereof (e.g., scFv).

[0203] As used herein, "human CD3" is defined as part of a multimolecular T cell receptor complex. CD3 represents an antigen expressed on human T cells, and CD3 has five different chains: CD3-ε, CD3 It consists of 3-γ, CD3-δ, CD3-η, and CD3-ζ.

[0204] For example, clustering of CD3 on T cells by anti-CD3 antibodies can lead to antigen binding. Similar, but it leads to T cell activation that is independent of the clonal specificity of the T cell subset. Therefore, one of its antigen-binding domains specifically binds to human CD3. The heavily specific molecule can bind to the human CD3 complex expressed on human T cells, and It can induce the removal / lysis of target cells, and such target cells are also bispecific molecules. The non-CD3 binding portion of the antigen (e.g., NPM1c:HLA-A2) is possessed / present This is demonstrated by a CD3-specific binder (e.g., the bispecific molecules disclosed herein). The binding of the CD3 complex results in the activation of T cells, which is known in the art; for example, international Pamphlet No. 99 / 54440 or International Pamphlet No. 2004 / 106381 See fret. In one embodiment, the bispecific molecule of the present disclosure is advantageously in v Target cells can be removed / lysed in vitro and / or iontophoretically. The target cells have tumor antigens on their surface, for example, bispecific molecules (i.e., bispecific NPM1c:H is recognized by another antigen-binding domain of the non-CD3 binding portion of the sex molecule. These may be cells that express or present LA-A2. In one embodiment, further specificity The sex is related to NPM1c:HLA-A2.

[0205] In one embodiment of the present disclosure, the V of the CD3-specific domain of a bispecific molecule H Region and V L The domains are OKT-3, X35-3, VIT3, BMA030 (BW264 / 56), C LB-T3 / 3, CRIS7, YTH12.5, F111409, CLB-T3.4.2 , TR-66, WT31, WT32, SPv-T3 b, 11D8, XIII-141, XIII46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW24B6 Selected from the group consisting of OKT3D, M-T301, SMC2, and F101.01. It is derived from CD3-specific antibodies. Each of these antibodies has been well described in the art. (For example, U.S. Patent No. 8007796 and U.S. Patent No. 884088) (See details).

[0206] In one embodiment, the CD3-specific domain of the bispecific molecule described herein is distributed At least one amino acid sequence including the sequence number 50 (YYDDHYCLDY) V H CDR3, indicated as sequence number 49 (YINPSRGYTNYNQKFKD) at least one V containing the amino acid sequence H CDR2, and / or Sequence ID 47 ( The amino acid sequence indicated as GYTFTRYTMH) or SEQ ID NO: 48(RYTMH) At least one V containing a column H Includes CDR1.

[0207] In some embodiments, the bispecific molecules of this disclosure have one or more CD3-specific domains. Multiple V L Includes region CDR. Such V of CD3-specific domains. L Area CDR is distributed At least one amino acid sequence including the sequence shown as column number 53 (QQWSSNPLT) V L CDR3, containing at least one amino acid sequence of sequence number 52 (DTSKVAS) V L As indicated by CDR2 and / or Sequence ID 51 (RASSSVSYMN) at least one V containing the amino acid sequence L Includes CDR1.

[0208] In one embodiment, the bispecific molecule is a single construct (e.g., C described herein). In DR, the V of antibodies that specifically bind to CD3 H and / or V L CDR1, C Includes DR2 and CDR3.

[0209] In one embodiment, the NPM1c:HLA-A2 specific domain of the bispecific molecule is The amino acid sequence shown in SEQ ID NO: 11 (ARLGYPTTTLLPFDY) is included in at least Both are one V H The CD3-specific domain of the bispecific molecule, which includes the CDR3 region, A small amount containing the amino acid sequence shown in sequence number 49 (YINPSRGYTNYNQKFKD) Even without one V H Includes the CDR2 region and / or Sequence ID 47 (GYTFTR The amino acid sequence includes at least the sequence shown in YTMH or SEQ ID NO: 48 (RYTMH). Another V H It includes the CDR1 region. In some embodiments, such a bispecific molecule , the V of each antigen-binding antibody L Further includes CDRs. For example, the NP of a bispecific molecule. The M1c:HLA-A2 specific domain is the amino acid sequence shown in Sequence ID No. 8 (QQSY At least one V including STPLT L CDR3 area, sequence number 7 (AAS) at least one V containing a noacid sequence L Includes the CDR2 area and / or the CD3 The specific domain is the amino acid sequence shown as Sequence ID No. 51 (RASSSVSYMN). at least one V including L Includes the CDR1 region. In one embodiment, as mentioned above. CDRs (e.g., CDR1, CDR2, CDR3) are contained in a single bispecific molecule. It can be done.

[0210] In one embodiment of this disclosure, the NPM1c:HLA-A2 and CD3 bispecific molecule is This disclosure includes both the heavy and light chains of anti-CD3 antibodies, CDR1, CDR2, and CDR3. In one embodiment, the NPM1c:HLA-A2 and CD3 bispecific molecule is an anti-NPM 1c: Contains both the heavy and light chains of the HLA-A2 antibody, CDR1, CDR2, and CDR3. Hmm. In some embodiments of this disclosure, the NPM1c:HLA-A2 and CD3 dual of this disclosure are used. The specific molecules are CDR1, CDR2, and CDR3 in both the heavy and light chains of the anti-CD3 antibody. , as well as CDR1 and CDR2 of both the heavy and light chains of anti-NPM1c:HLA-A2 antibodies and includes CDR3.

[0211] In one embodiment, the anti-CD3 CDR referred to herein is Kabat numbering system Follow the system. The Kabat numbering scheme is a consistent way of numbering residues in antibodies. This is a widely adopted standard for labeling (Kabat et al., Sequences of Proteins of Immunological Interest, 1991. This is incorporated herein in its entirety by reference. ).

[0212] In one embodiment of this disclosure, the NPM1c:HLA-A2 and CD3 bispecific molecule is (a) NPM1c:HLA-A2 heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 5 Area(V H ); and / or NPM1c:HL containing the amino acid sequence shown in SEQ ID NO: 3 A-A2 light chain variable region (V L ); Furthermore (b) CD3-specific antibodies (e.g., OKT-3, X35-3, VIT3, BMA030) (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-4 09, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141 , XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2- Select from the group consisting of 4B6, OKT3D, M-T301, SMC2, and F101.01. CD3 heavy chain variable region (VH) and / or light chain variable region (VL) derived from the antibody (which is used to obtain the antibody) Includes.

[0213] In one embodiment of this disclosure, the NPM1c:HLA-A2 and CD3 bispecific molecule is (a) NPM1c:HLA-A2 heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 5 Area(V H ); and NPM1c:HLA-A2 containing the amino acid sequence shown in Sequence ID No. 3 Light chain variable region (V L ); Furthermore (b) CD3-specific antibodies (e.g., OKT-3, X35-3, VIT3, BMA030) (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-4 09, TR-66, WT31, WT32, SPv-T3b, 11D8, XIII-141 , XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2- Select from the group consisting of 4B6, OKT3D, M-T301, SMC2, and F101.01. CD3 heavy chain variable region (VH) and light chain variable region (VL) derived from the antibody (which is used) Includes.

[0214] Natural killer (NK) cell activity includes both activating and repressing signals. It is regulated by a complex mechanism. NK cell-mediated recognition of HLA class I-deficient target cells. And several different NK cell receptors that play an important role in killing have been identified. Yes. One of the receptors is not specific to NK cells, but FcγRIIIA(CD16A) This is involved in NK cell-mediated cytotoxicity (ADCC). Another NK cell receptor is It is NKp46, a member of the Ig superfamily. This is specific to NK cells. Therefore, the crosslinking induced by specific mAbs leads to strong NK cell activation, This leads to an increase in intracellular Ca++ levels, induction of cytotoxicity, and release of lymphokines. .

[0215] In some embodiments, the bispecific molecules of this disclosure (e.g., bispecific antibodies or fragments) NPM1c: simultaneously binds to HLA-A2 and NKp46. In some embodiments, this The disclosed bispecific molecule is NPM1c:HLA-A2 expressed or presented on tumor cells. And it simultaneously binds to NKp46 expressed on NK cells. In some embodiments, this two The heavily specific molecule is a single-chain variable (scFv) fragment that recognizes NPM1c:HLA-A2, and It contains an antibody or antigen-binding fragment (e.g., scFv) that binds to NKp46. Antibodies and / or antigen-binding fragments that specifically bind to p46 are known in the art. (For example, International Publication No. 15 / 197593, International Publication No. 17 / 11469) (See pamphlet No. 4).

[0216] In some embodiments, the bispecific molecules of this disclosure (e.g., bispecific antibodies or fragments) It binds simultaneously to NPM1c:HLA-A2 and CD16A. In some embodiments, The bispecific molecules of this disclosure are expressed or presented on tumor cells, and NPM1c:HLA- It simultaneously binds to CD16A expressed on A2 and NK cells. In some embodiments, This bispecific molecule is a single-chain variable (scFv) fragment that recognizes NPM1c:HLA-A2. and includes its antibody or antigen-binding fragment (e.g., scFv) that binds to CD16A. Antibodies and / or antigen-binding fragments that specifically bind to CD16A are known in the art. (For example, Stein et al., (2012) Antibodies 1:88-123, and cited therein) (See references.)

[0217] In some embodiments, the binding of the bispecific molecules provided in this disclosure to NK cells NK cells are further activated. In some embodiments, the dual features provided by this disclosure The binding of isomeric molecules to NK cells induces the antitumor activity of NK cells. In this state, the binding of the bispecific antibody provided by this disclosure to NK cells leads to antibody-dependent Sex cell-mediated cytotoxicity (ADCC) is induced.

[0218] Therefore, in some embodiments, this disclosure is, (i) Class I major histocompatibility complex (MHC class I) proteins (e.g., HLA- The first antigen binding specifically binds to the NPM1c neoepitope that forms a complex with A2). domain; and (ii) The following: CD3, NKp46, CD16A, CD40, CD47, 4-1 BB, TGF-β, LAG-3, PD-1, TIM-3, CTLA-4, OX-40, N It specifically binds to one of the following: Kp30, NKG2A, NKG2D, or DNAM-1. Second antigen-binding domain The present invention provides a bispecific antigen-binding polypeptide containing the present invention.

[0219] In some embodiments, this disclosure is, (i) Class I major histocompatibility complex (MHC class I) proteins (e.g., HLA- The first antigen binding specifically binds to the NPM1c neoepitope that forms a complex with A2). domain; and (ii) A second antigen-binding domain that specifically binds to CD3 (e.g., human CD3) The present invention provides a bispecific antigen-binding polypeptide containing a second antigen-binding polypeptide. In some embodiments, the second antigen-binding polypeptide contains The synthesis domain specifically binds to human CD3 expressed on T cells.

[0220] In some embodiments, this disclosure is, (i) Class I major histocompatibility complex (MHC class I) proteins (e.g., HLA- The first antigen binding specifically binds to the NPM1c neoepitope that forms a complex with A2). domain; and (ii) A second antigen-binding agent that specifically binds to NKp46 (e.g., human NKp46) main The present invention provides a bispecific antigen-binding polypeptide containing a second antigen-binding polypeptide. In some embodiments, the second antigen-binding polypeptide contains The synthetic domain specifically targets human NKp46 expressed on natural killer (NK) cells. Combine.

[0221] In some embodiments, this disclosure is, (i) Class I major histocompatibility complex (MHC class I) proteins (e.g., HLA- The first antigen binding specifically binds to the NPM1c neoepitope that forms a complex with A2). domain; and (ii) A second antigen-binding agent that specifically binds to CD16A (e.g., human CD16A) main The present invention provides a bispecific antigen-binding polypeptide containing a second antigen-binding polypeptide. In some embodiments, the second antigen-binding polypeptide contains The synthesis domain specifically binds to human CD16A expressed on NK cells.

[0222] In some embodiments, the NPM1c neoepitope is an amino acid selected from the following: Contains acid sequences: AIQDLCVAV (SEQ ID NO: 71), CLAVEEVSL (SEQ ID NO: 71) 2) VEEVSLRK (sequence number 73), AVEEVSLR (sequence number 74), AVE EVSLRK (sequence number 75) and CLAVEEVSLRK (sequence number 76). One implementation Morphologically, the NPM1c neoepitope has the amino acid sequence AIQDLCLAV (SEQ ID NO: 1). ) includes.

[0223] In some embodiments, MHC class I proteins belong to the HLA-A*02 allele group. Encoded by an HLA-A allele containing . In some embodiments, the HLA-A allele This is HLA-A*02:01.

[0224] In some embodiments, the first antigen-binding domain is a heavy chain variable region (VH) and / or VH contains the light chain variable region (VL), and VH contains the amino acid sequence shown in SEQ ID NO: 5. It contains the amino acid sequence shown in Sequence ID No. 3.

[0225] In some embodiments, the first antigen-binding domain is a heavy chain variable region (VH) and / or It includes the light chain variable region (VL), and VH is at least the amino acid sequence shown in SEQ ID NO: 5. 90%, at least 95%, at least 98%, or at least 99% identical amino acids The sequence includes, and VL is at least 90% and at least the amino acid sequence shown in SEQ ID NO: 3. They also contain 95%, at least 98%, or at least 99% identical amino acid sequences.

[0226] In some embodiments, the first antigen-binding domain is a heavy chain variable region (VH) and a light chain variable region. It includes a variable region (VL), VH includes the amino acid sequence shown in SEQ ID NO: 5, and VL is distributed It includes the amino acid sequence shown in column number 3. In some embodiments, the first antigen-binding domain It includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH is shown in Sequence ID No. 5. The amino acid sequence is at least 90%, at least 95%, at least 98%, or less It contains at least 99% identical amino acid sequences, and VL is the amino acid sequence shown in SEQ ID NO: 3. and are at least 90%, at least 95%, at least 98%, or at least 99% identical. It contains a specific amino acid sequence.

[0227] In some embodiments, the first antigen-binding domain is sequence numbers 9, 10, and 1, respectively. The VH CDR1, VH CDR2, and VH CDR3 sequences shown in 1, and / or or VL CDR1, VL CDR2 and shown in sequence numbers 6, 7, and 8 respectively. Contains VL CDR3 sequence

[0228] In some embodiments, the first antigen-binding domain is sequence numbers 9, 10, and 1, respectively. The VH CDR1, VH CDR2, and VH CDR3 sequences shown in 1, and their VL CDR1, VL CDR2, and VL CDR1 are shown in sequence numbers 6, 7, and 8, respectively. Includes the DR3 sequence.

[0229] In some embodiments, the first antigen-binding domain is scFv, Fab, or F(ab It includes ')2, and the second antigen-binding domain is scFv, and Fab, or F(ab' ) Includes 2.

[0230] The antibodies or their corresponding immunoglobulin chains used in accordance with this disclosure are subject to this technology. Using conventional methods known in the field, for example, amino acid deletion, insertion, substitution, addition, and / Alternatively, recombination and / or any other modifications known in the art, either alone or in combination. It can be further modified by using any of the following: Immunoglobulin chain Methods for introducing such modifications into the DNA sequence that forms the basis of the amino acid sequence are available to our company. This is common knowledge among those who know; for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold S See Pring Harbor Laboratory (1989) NY. The modifications mentioned are preferably at the nucleic acid level. It will be held at [location].

[0231] Bispecific antibodies are produced by various methods, including hybridoma fusion or Fab' fragment linking. It can be produced by, for example, Songsivilai & Lachmann, Clin. Exp. Immunol See 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Traditionally, the recombinant production of bispecific antibodies involves the simultaneous development of two immunoglobulin heavy / light chain pairs. Based on the current model, the two heavy / light chain pairs have different specificities (Milstein and Cuell). o, (1983) Nature 305:537-539). Antibodies with desired binding specificity (antibody-antigen binding site). The variable domain can be fused to the constant domain sequence of immunoglobulins. The fusion of the variable regions preferably includes at least a portion of the hinge, CH2, and CH3 regions. This involves fusion with the constant domain of the immunoglobulin heavy chain. This is necessary for generating bispecific antibodies. For further details on the exemplary, currently known method, see, for example, Suresh et al., (1986). Methods Enzymol. 121:210; PCT Publication No. WO 96 / 27011; Brennan et al., (1985 ) Science 229:81; Shalaby et al., J. Exp. Med. (1992) 175:217-225; Kostelny et a l., (1992) J. Immunol. 148(5):1547-1553; Hollinger et al., (1993) Proc. Natl. Ac ad. Sci. USA 90:6444-6448; Gruber et al., (1994) J. Immunol. 152:5368; and Tutt See et al., (1991) J. Immunol. 147:60. For bispecific antibodies, cross-linked antibodies or heterochromic antibodies are used. This also includes loconjugate antibodies. Heteroconjugate antibodies are a convenient cross-linking method. It can be manufactured using [a suitable crosslinking agent]. Suitable crosslinking agents are well known in the art and in the United States. It is disclosed in Patent No. 4,676,980, along with numerous crosslinking methods.

[0232] Various methods for directly producing and isolating bispecific antibody fragments from recombinant cell cultures. It is also stated that, for example, bispecific antibodies are prepared using a leucine zipper. Yes. For example, see Kostelny et al. (1992) J Immunol 148(5):1547-1553. The leucine zipper peptide derived from Jun protein undergoes two different processes through gene fusion. It can be linked to the Fab' portion of the antibody. The antibody homodimer is reduced at the hinge region. It can form a nomer, and then be reoxidized to form an antibody heterodimer. The method can also be used for the production of antibody homodimers. Hollinger et al. (1993) The "diabody" technique described by Proc Natl Acad Sci USA 90:6444-6448 is two It provides an alternative mechanism for producing heavily specific antibody fragments. These fragments are on the same chain. The linker is too short to allow matching between the two domains, resulting in a light chain variable domain ( It contains a heavy chain variable domain (VH) connected to the VL. Therefore, the VH of one fragment The VL domain is forced to align with the complementary VL and VH domains of another fragment, This results in the formation of two antigen-binding sites. By using a single-chain Fv(scFv) dimer... Other strategies for producing bispecific antibody fragments have also been reported. For example, Gruber et al. See (1994) J Immunol 152:5368. Alternatively, for antibodies, see, for example, Zapata et al. (1995 ) It may be a "linear antibody" as described in Protein Eng. 8(10):1057-1062. In short, these antibodies form a pair of tandem Fd-segments that combine a pair of antigen-binding regions. Contains ment (VH-CH1-VH-CH1). Linear antibodies are bispecific or monospecific. It could be someone of the opposite sex.

[0233] This disclosure also describes variant forms of multispecific antibodies, e.g., Wu et al. (2007) Nat Bio The dual variable domain immunoglobulin described in technol 25(11): 1290-1297 (DVD It also includes the -Ig) molecule. The DVD-Ig molecule is derived from two different parent antibodies. Light chain variable domains (VLs) are acquired by recombinant DNA technology, either directly or via short linkers. They are designed to be linked in tandem, and then the light chain constant domain is linked in a similar manner. The heavy chain consists of two different heavy chain variable domains (VHs) linked in tandem, followed by... It contains the constant domain CH1 and Fc region. DVD-Ig molecules are produced from two parent antibodies. For example, the method for doing so is described in PCT Public International Publication No. 08 / 024188 and Further details are provided in International Publication No. 07 / 024715. Some implementations In this state, a bispecific antibody has a light chain variable region with a second specificity that is located within the heavy chain variable region of the entire antibody. It is a Fab-in-Tandem immunoglobulin fused to the region. Such an antibody For example, as described in the brochure for International Patent Application Publication No. 2015 / 103072 ru.

[0234] Chimeric antigen receptor In one embodiment, a chimeric antigen receptor (CAR) including an extracellular domain, The ink is any of the antibodies described herein, or their antigen-binding fragments, or duplexes. CARs comprising specific molecules are provided herein. In certain embodiments, The chimeric antigen receptor (CAR) provided in the specification is a neoantigen (e.g., cancer neoantigen) It contains an extracellular domain that binds to tumor neoantigens. Cancer neoantigens are formed when cancer cells are affected. Due to a mutation, it is an antigen that exists only in such cancer cells. Cancer antigens are found within cells. It can be expressed and presented on the surface of cancer cells by MHC class I proteins, for example. The cancer neoantigen targeted by CAR as envisioned herein is NPM1c:HL It may be A-A2. In certain embodiments, the antibody or antigen-binding fragment of the present disclosure (e.g.) Chimeric antigen receptors (CARs) can be manufactured using scFv. In the application form, an antibody or antigen-binding fragment that binds to NPM1c:HLA-A2 (for example) Using scFv), chimeric antigen receptor (CAR) polypeptides are generated. In certain embodiments, the chimeric antigen receptor (CAR) includes an extracellular binding domain, The extracellular binding domain cleaves the binding of any of the antibodies described herein or their antigens. A fragment or bispecific molecule, such an antibody, its antigen-binding fragment, or bispecific Sex molecules are Class I major histocompatibility complex (MHC class I) proteins (e.g., HLA). -2) Mutant nucleophosmin that forms a complex with (or is presented thereby with) CARs that bind to protein neoepitopes (e.g., NPM1c neoepitope) Provided herein.

[0235] CARs are genetically engineered artificial membrane-binding proteins used in immune effector cells. When expressed in such a state, such immune effector cells are directed to the antigen, and generally, immunity It stimulates effector cells to kill antigen-presenting cells. Therefore, chimeric anti Using protocortical receptors (CARs), immune effector cells can be given desired antigen specificity, for example, anti- Tumor specificity can be conferred (in particular, antigen specificity is determined by the extracellular domain of the CAR). (It is granted.)

[0236] CARs generally have an extracellular domain that binds to one or more antigens presented on a cell. The transmembrane domain and the extracellular domain are activated when they bind to one or more antigens. Includes an intracellular domain that transmits the signal to immune effector cells. In certain embodiments Therefore, CAR contains the following three domains: 1) Typically a signal peptide, A cell containing a ligand or antigen recognition region (e.g., scFv) and a mobile spacer. 1) Outer domain; 2) Transmembrane (TM) domain; 3) Typically one or more signaling domains The intracellular domain (also known as the cytoplasmic domain) containing the cytoplasmic domain. The extracellular domain of CAR. The domain is located outside the cell and is exposed to the extracellular space, therefore its ligand / antigen It is accessible for interaction with the TM domain. The TM domain is accessible to the effector cell. The intracellular domain of the CAR allows it to be anchored to the cell membrane. One or more signaling proteins derived from a protein different from the original protein. It may contain a cytoplasmic domain. The intracellular domain is where the CAR is bound to its ligand / antigen. In this process, it helps activate effector cells. In some embodiments, effector cell activation is , induction of cytokine and chemokine production, and cytolytic activity of effector cells This includes sexual activation. In some embodiments, CAR redirects cytotoxicity to tumor cells. Delete.

[0237] The engagement between the antigen-binding domain of a CAR and its target antigen on the surface of the target cell is This leads to CAR clustering and delivers activation stimuli to CAR-containing cells. In terms of application methods, the main characteristic of CAR is that it redirects the specificity of immune effector cells, This leads to proliferation, cytokine production, phagocytosis, or monoclonal antibodies, Major groups that utilize the cell-specific targeting ability of soluble ligands or cell-specific coreceptors Molecules that can mediate cell death of target antigen-expressing cells in a manner independent of tissue compatibility complexes (MHCs). It is the ability to induce production. The signaling domain from CD3ζ or FcRy. CARs based on scFv, which have been manipulated to contain [specific component], are used for T cell activation and effector —It has been shown to deliver strong signals for function, but there are no co-stimulatory signals. In some cases, it may not be sufficient to induce signals that promote T cell survival and proliferation. Yes, it has a binding domain, hinge, transmembrane domain, and is derived from CD3ζ or FcRy. The signaling domain is one or more co-stimulatory signaling domains (e.g., C (Intracellular costimulatory domains derived from D28, CD137, CD134, and CD278) The new generation of CARs contained in both exhibits more effective antitumor activity in CAR-expressing T cells. In addition to leading to increased cytokine secretion, lytic activity, survival, and proliferation. This has been demonstrated in vitro, in animal models, and in cancer patients (Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al., PNAS, 2009; 106:3360-3365).

[0238] In some embodiments, the extracellular (antigen-binding) domain, the transmembrane domain, and the antigen-binding domain The cytoplasmic sequence of CD3ζ contains enough CD3ζ sequences to stimulate T cells when the antigen is bound to it. In addition to the intracellular (cytoplasmic) domain, optionally, the antigen-binding domain binds to the antigen. In some cases, one or more (e.g., two, three, or four) T cells co-stimulate T cells. The cytoplasmic sequence of the co-stimulatory protein (e.g., B7-H3, BTLA, CD2, CD7, CD27, CD28, CD30, CD40, CD40L, CD80, CD160, CD2 44, ICOS, LAG3, LFA-1, LIGHT, NKG2C, 4-1BB, OX4 0, PD-1, PD-L1, TIM3, 2B4, DAP10, CD137, DAP12, (and one or more cytoplasmic sequences of ligands that specifically bind to CD83) A CAR is provided herein. In some embodiments, the CAR is a linker It may also include: exemplary extracellular (antigen-binding) domains, linkers, transmembrane domains, etc. CARs and CAR-expressing immunoeffector cells, including intracellular (cytoplasmic) domains. Further aspects can be seen, for example, in Kakarla et al., Cancer J. 20:151-155, 2014; Srivastava e t al., Trends Immunol. 36:494-502, 2015; Nishio et al., Oncoimmunology 4(2): e98 8098, 2015; Ghorashian et al., Br. J. Haematol. 169:463-478, 2015; Levine, Cance r Gene Ther. 22:79-84, 2015; Jensen et al., Curr. Opin. Immunol. 33:9-15, 2015; Singh et al., Cancer Gene Ther. 22:95-100, 2015; Li et al., Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1753-1756, 2014; Gill et al., Immunol. Rev. 263:68-89, 2015; M agee et al., Discov. Med. 18:265-271, 2014; Gargett et al., Front. Pharmacol. 5: 235, 2014; Yuan et al., Zhongguo Shi Yan Xue Ye Xue Za Zhi 22:1137-1141, 2014; P edgram et al., Cancer J. 20:127-133, 2014; Eshhar et al., Cancer J. 20:123-126, 2014; Ramos et al., Cancer J. 20:112-118, 2014; Maus et al., Blood 123:2625-2635 , 2014; Jena et al., Curr. Hematol. Malig. Rep. 9:50-56, 2014; Maher et al., Cur r. Gene Ther. 14:35-43, 2014; Riches et al., Discov. Med. 16:295-302, 2013; Chea dle et al., Immunol. Rev. 257:83-90, 2014; Davila et al., Int. J. Hematol. 99:36 1-371, 2014; Xu et al., Cancer Lett. 343:172-178, 2014; Kochenderfer et al., Nat . Rev. Clin. Oncol. 10:267-276, 2013; Hosing et al., Curr. Hematol. Malig. Rep. 8:60-70, 2013; Hombach et al., Curr. Mol. Med. 13:1079-1088, 2013; Xu et al., Le uk. Lymphoma 54:255-260, 2013; Gilham et al., Trends Mol. Med. 18:377-384, 2012; Lipowska-Bhalla et al., Cancer Immunol. Immunother. 61:953-962, 2012; Chmielews ki et al., Cancer Immunol. Immunother. 61:1269-1277, 2013; Jena et al., Blood 11 6:1035-1044, 2010; Dotti et al, Immunology Reviews 257(1): 107-126, 2013; Dai et al al., Journal of the National Cancer Institute 108(7): djv439, 2016; Wang and Ri viere, Molecular Therapy-Oncolytics 3: 16015, 2016; US Patent Application No. 2018 / Document No. 0057609; same as Document No. 2018 / 0037625; same as Document No. 2017 / Invoice No. 0362295; same as Invoice No. 2017 / 0137783; same as Invoice No. 2016 / 0152 Invoice No. 723, Invoice No. 2016 / 0206656, Invoice No. 2016 / 0199412 Specifications, including Specification No. 2016 / 0208018 and Specification No. 2015 / 0232880. Same as Specification No. 2015 / 0225480; Same as Specification No. 2015 / 0224143; Same as 201 Invoice No. 5 / 0224142; same as Invoice No. 2015 / 0190428; same as Invoice No. 2015 / 01 Invoice No. 96599; same as Invoice No. 2015 / 0152181; same as Invoice No. 2015 / 014002 Item No. 3; same as Item No. 2015 / 0118202; same as Item No. 2015 / 0110760 Book; same as item number 2015 / 0099299; same as item number 2015 / 0093822; same as 2 Inventory No. 015 / 0093401; same as Inventory No. 2015 / 0051266; same as 2015 / Inventory No. 0050729; same as Inventory No. 2015 / 0024482; same as 2015 / 0023 Item No. 937; same as Item No. 2015 / 0017141; same as Item No. 2015 / 0017136 Inventory; same as Inventory No. 2015 / 0017120; same as Inventory No. 2014 / 0370045; Same as Detailed Statement No. 2014 / 0370017; Same as Detailed Statement No. 2014 / 0369977; Same as 201 Item No. 4 / 0349402; same as Item No. 2014 / 0328812; same as Item No. 2014 / 03 Item No. 22275; same as Item No. 2014 / 0322216; same as Item No. 2014 / 032221 Item No. 2; same as Item No. 2014 / 0322183; same as Item No. 2014 / 0314795 Book; same as item number 2014 / 0308259; same as item number 2014 / 0301993; same as 2 Item No. 014 / 0296492; same as Item No. 2014 / 0294784; same as 2014 / Item No. 0286973; same as Item No. 2014 / 0274909; same as Item No. 2014 / 0274 Inventory No. 801; same as Inventory No. 2014 / 0271635; same as Inventory No. 2014 / 0271582 Inventory; same as Inventory No. 2014 / 0271581; same as Inventory No. 2014 / 0271579; Same as Detail Sheet No. 2014 / 0255363; Same as Detail Sheet No. 2014 / 0242701; Same as 201 Item No. 4 / 0242049; same as Item No. 2014 / 0227272; same as Item No. 2014 / 02 Inventory No. 19975; same as Inventory No. 2014 / 0170114; same as 2014 / 013472 Specification No. 0; Specification No. 2014 / 0134142; Specification No. 2014 / 0120622 Book; 2014 / 0120136 specification; 2014 / 0106449 specification; 2 014 / 0106449 specification; 2014 / 0099340 specification; 2014 / 0086828 specification; 2014 / 0065629 specification; 2014 / 0050 Specification No. 708; Specification No. 2014 / 0024809; No. 2013 / 0344039 Specification; 2013 / 0323214 specification; 2013 / 0315884 specification; 2013 / 0309258 specification; 2013 / 0288368 specification; 201 3 / 0287752 specification; 2013 / 0287748 specification; 2013 / 02 80221 specification; 2013 / 0280220 specification; 2013 / 026655 Specification No. 1; Specification No. 2013 / 0216528; Specification No. 2013 / 0202622 Book; 2013 / 0071414 specification; 2012 / 0321667 specification; 2 012 / 0302466 specification; 2012 / 0301448 specification; 2012 / 0301447 specification; 2012 / 0060230 specification; 2011 / 0213 Specification No. 288; Specification No. 2011 / 0158957; Specification No. 2011 / 0104128 Specification; 2011 / 0038836 specification; 2007 / 0036773 specification; and as described in the same specification No. 2004 / 0043401. Exemplary extracellular (antigen) CA includes a combined domain, linker, transmembrane domain, and intracellular (cytoplasmic) domain. Further aspects of R and CAR-expressing immunoeffector cells are described in International Publication No. 2016 / 16. Pamphlet No. 8595; International Publication No. 12 / 079000 Pamphlet; Same as above, 2015 / Pamphlet No. 0141347; Pamphlet No. 2015 / 0031624; Pamphlet No. 201 Pamphlet No. 5 / 0030597; Pamphlet No. 2014 / 0378389; Pamphlet No. 2 Pamphlet No. 014 / 0219978; Pamphlet No. 2014 / 0206620; Pamphlet No. 2014 / 0037628; Pamphlet No. 2013 / 0274203 T; Pamphlet No. 5668, 2013 / 02 / 2013; Pamphlet No. 16167, 2013 / 01 / 10 Let's; Pamphlet No. 962, 2012 / 0230; Pamphlet No. 783, 2012 / 0213 Brochure; Pamphlet No. 2012 / 0093842; Pamphlet No. 2012 / 0071420 Pamphlet No. 2012 / 0015888; Pamphlet No. 2011 / 02687 Pamphlet No. 54; same 2010 / 02 / 97093; same 2010 / 015 Pamphlet No. 8881; Same as 2010 / 0034834; Same as 2010 / 0 Pamphlet No. 015113; same 2009 / Pamphlet No. 0304657; same 2004 Pamphlet No. 0043401; Pamphlet No. 2014 / 0322253; Pamphlet No. 20 Pamphlet No. 15 / 0118208; Pamphlet No. 2015 / 0038684; Same Pamphlet No. 2014 / 0024601; Pamphlet No. 2012 / 0148552 ; Pamphlet No. 3129, 2011 / 022; Pamphlet No. 7994, 2009 / 025 Pamphlet No. 2008 / 0160607; Bread No. 2008 / 0003683 Fret; Pamphlet No. 2013 / 0121960; Pamphlet No. 2011 / 0052554 This information is included in the pamphlet and in pamphlet number 2010 / 0178276.

[0239] In some embodiments, CARs include an intracellular domain, a transmembrane domain, and an extracellular domain. The extracellular domain is one of the antibodies or antigens described herein. CARs comprising binding fragments or bispecific molecules are provided herein. In one embodiment, a chimeric antigen comprising an intracellular domain, a transmembrane domain, and an extracellular binding domain. A receptor (CAR) whose extracellular binding domain is any of the anti- The body, or its antigen-binding fragment, or bispecific molecule, and such antibodies, their anti- The proto-binding fragment, or bispecific molecule, is a Class I major histocompatibility complex (MHC Class I). NPM1c neoepidopsy, which forms a complex with (or is presented by) the protein A CAR that binds to an antigen containing a loop is provided herein.

[0240] In some embodiments, the intracellular and transmembrane NPM1c CARs described in the Examples section are used. and / or chimeric antigen receptors (CARs) having an extracellular domain, as specified herein It is provided as shown (see, for example, Example 3).

[0241] In some embodiments, the following: CD27, CD28, 4-1BB, OX40, CD30, CD 40, PD-1, ICOS, 2B4, DAP10, CD137 and DAP12, Select from the group, one or more co-stimulatory domains of one or more co-stimulatory molecules CARs containing intracellular domains are provided herein. In certain embodiments, The CD3-zeta signaling domain and, optionally, the 4-1BB costimulatory domain A CAR containing an intracellular domain is provided herein. In some embodiments, C D3-Zeta, CD8, CD28, NKG2D, CD16, NKp44, or NKp46 A CAR comprising a transmembrane domain is provided herein. In certain embodiments, A CAR containing a transmembrane domain including a CD8 transmembrane domain is provided herein. In some embodiments, any antibody or antigen-binding fragment described herein (e.g.) For example, a CAR containing an extracellular domain including scFv is provided herein. In certain embodiments, class I major histocompatibility complex (MHC class I) proteins (for example) For example, a mutant nucleus that forms a complex with (or is presented by) HLA-A2. Antigens containing an ophosmin protein epitope (e.g., NPM1c neoepitope) Any antibody or antigen-binding fragment described herein that specifically binds (e.g. In this specification, a CAR containing an extracellular domain including scFv is provided. In certain embodiments, any of the antibodies described herein, including VH and VL, or A CAR comprising an extracellular domain containing the antigen-binding fragment (e.g., scFv), V H is at least 75%, 80%, and 85% of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 5 , containing 90%, 95%, 98%, or 99% identical amino acid sequences, VL is the sequence number. 3 amino acid sequence or SEQ ID NO: 3 and at least 75%, 80%, 85%, 90%, 95% CARs containing %, 98%, or 99% identical amino acid sequences are provided herein. In a particular embodiment, VH CDR1 having the amino acid sequence of sequence number 9, sequence number VH CDR2 has the amino acid sequence of sequence 10, and C has the amino acid sequence of sequence number 11. VH containing DR3 and / or VL C having the amino acid sequence of SEQ ID NO: 6 VL CDR2 having the amino acid sequence of DR1, SEQ ID NO: 7, and amino acid sequence of SEQ ID NO: 8 Any antibody described herein, comprising a VL containing a VL CDR3 having an acid sequence Alternatively, a CAR containing an extracellular domain that includes its antigen-binding fragment (e.g., scFv) is a CAR that Provided in the specification. In a particular embodiment, V having the amino acid sequence of SEQ ID NO: 5 VH includes VH CDR1, VH CDR2, and VH CDR3, which are H-rated CD-Rs. VL CD, which is a CDR of VL having the amino acid sequence of SEQ ID NO: 3. VLs including R1, VL CDR2 and VL CDR3 as described herein It includes an extracellular domain containing either an antibody or its antigen-binding fragment (e.g., scFv). CAR is provided herein. In a particular embodiment, the amino acid combination of SEQ ID NO: 2 scFv having a column, or array index 2 and at least 75%, 80%, 85%, 90%, Extracellular domains containing scFv with 95%, 98%, or 99% identical amino acid sequences A CAR containing n is provided herein.

[0242] Examples of extracellular, transmembrane, and intracellular domains of CARs provided herein are as follows: See below.

[0243] Antibody-antigen binding domain, including the extracellular (antigen-binding) domain of CAR Non-exclusive examples of antigen-binding domains include: monoclonal Antibodies (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgE, and Ig D) Antigen binding cleavage of antibodies (e.g., fully human antibodies or chimeric (e.g., humanized) antibodies). Fragments (e.g., Fab, Fab', or F(ab')2 fragments) (e.g., fully human antibodies) Chimeric (e.g., humanized) antibody fragments, diabodies, triabodies, tetrabodies i, minibody, scFv, scFv-Fc, (scFv)2, scFab, bis-s cFv, hc-IgG, BiTE, single-domain antibody (e.g., V-NAR domain or (VhH domain), IgNAR, and multispecific (e.g., bispecific antibody) antibodies. In one embodiment, the antigen-binding domain includes scFv. These antigen-binding domains are manufactured The method is well known in the art.

[0244] In some embodiments, the antigen-binding domain is an antibody that can specifically bind to a target antigen, for example For example, immunoglobulin molecules (e.g., light chain or heavy chain immunoglobulin molecules) and immunoglobulins At least one (e.g., one, two) immunoactive (antigen-binding) fragment of a brin molecule CDRs (e.g., 3, 4, 5, or 6) immunoglobulin light chain variable domains One of the three CDRs of origin, and / or the immunoglobulin heavy chain variable domain. Includes one of the three CDRs from which it originated.

[0245] In some embodiments, the antigen-binding domain is a single-chain antibody (for example, the V-NAR domain is also included). or V H It is either an H domain or one of the single-chain antibodies described herein. In this embodiment, the antigen-binding domain is the entire antibody molecule (e.g., human antibody, humanized antibody, These are either chimeric antibodies or multimer antibodies (e.g., bispecific antibodies).

[0246] In some embodiments, the antigen-binding domain contains an antibody fragment and multispecificity (for example, two This includes (heavily specific) antibodies or antibody fragments. Examples of antibodies and their antigen-binding fragments include single-chain antibodies. Fv(scFv), Fab fragment, Fab' fragment, F(ab')2, disulfide-linked Fv (sdFv), Fv, and a fragment containing either the VL domain or the VH domain. This includes, but is not limited to, the following:

[0247] Further antigen-binding domains provided herein may be polyclonal or monoclonal. Human antibodies, multispecific (multimer, e.g., bispecific), chimeric antibodies (e.g.) For example, human-mouse chimeras, single-chain antibodies, antibodies produced within cells (i.e., intra Antibodies (bodies) and their antigen-binding fragments. Antibodies or their antigen-binding fragments are any Type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g.) (For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or s It may be a buclas. In some embodiments, the antigen-binding domain is an IgG1 antibody This is the antigen-binding fragment. In some cases, the antigen-binding domain is the IgG4 antibody or This is the antigen-binding fragment. In some embodiments, the antigen-binding domain consists of the heavy chain and the light chain. It contains immunoglobulins.

[0248] Further examples of antigen-binding domains include antigen-binding fragments of IgG (e.g., IgG1 2. Antigen-binding fragments of IgG3 or IgG4) (e.g., human or humanized IgG, e.g.) For example, antigen binding cleavage of human or humanized IgG1, IgG2, IgG3, or IgG4. (A fragment), an antigen-binding fragment of IgA (for example, an antigen-binding fragment of IgA1 or IgA2) (for example) If human or humanized IgA, for example, human or humanized IgA1 or IgA2 antibodies (Original binding fragment), IgD antigen-binding fragment (e.g., human or humanized IgD antigen-binding fragment) ), antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE), or It is an antigen-binding fragment of IgM (for example, an antigen-binding fragment of human or humanized IgM).

[0249] In some embodiments, the antigen-binding domain is, for example, in saline or phosphate-buffered saline. Within that, for a specific antigen (e.g., tumor-associated antigen), approximately 1 × 10⁻⁶ -7 M or stronger (for example) Then, approximately 1 x 10 -8 M or stronger, approximately 1 x 10 -9 M or stronger, approximately 500 nM or stronger, approximately 100nM or stronger, approximately 25nM or stronger, Approximately 15 nM or stronger, approximately 7 nM or stronger, approximately 5 nM or weaker, The affinity (K) is approximately 1 nM or stronger. D They can be joined together using ).

[0250] As those skilled in the art will understand, the selection of antigen-binding domains to be included in CAR is Define the surface of the target cells (e.g., cancer cells or tumors) in the required area. It depends on the type and number of ligands used. For example, the antigen-binding domain is used with tumor-specific antigens. TSAs may be selected to recognize cancer neoantigens, for example, tumor-specific antigens. Antigens include MHC class I proteins (e.g., HLA-A2), e.g., NPM1c:H NMP1c neoantigen that has formed a complex with (or is presented by) LA-A2 It may be present. In some embodiments, the NMP1c neoantigen has the amino acid sequence AIQDLC Includes LAV (sequence number 1).

[0251] In some embodiments, the CAR molecule is an antigen-binding molecule that recognizes tumor antigens of acute myeloid leukemia. Includes a domain. In some embodiments, the tumor antigen is a tumor-specific antigen (TSA), for example. TSA is an acute myeloid leukemia neoantigen. TSA is specific to tumor cells and not to other cells in the body. It does not exist above. In one embodiment, the tumor antigen is a tumor-specific antigen. In a particular implementation Morphologically, tumor-specific antigens sequence the patient's tumor cells and are mutations found only in tumors. This is determined by identifying different proteins. These antigens are called "neoantigens" and It is called [a specific term]. Once a neoantigen is identified, it is used to induce the production of therapeutic antibodies against it. It can be used in the method described in the specification. In one embodiment, the neoantigen is NPM1 The c neoantigen is an MHC class I protein. In one embodiment, the NMP1c neoantigen is an MHC class I protein. (For example, HLA-A2), for example, NPM1c: forms a complex with HLA-A2. (or as presented therein).

[0252] Tumor antigens that can be targeted by CAR effector cells (e.g., CAR T cells) For example, tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs) include NPM1c: This includes, but is not limited to, HLA-A2. In one embodiment, the tumor-specific antigen is NPM1c:HLA-A2

[0253] CAR's inter-domain linker The linker can be optionally placed between (1) the extracellular (antigen-binding) domain and the transmembrane domain. (2) CARs that may be included between the transmembrane domain and the intracellular (cytoplasmic) domain , provided herein. In some embodiments, the linker is a polypeptide linker. —This is possible. For example, a linker is made up of approximately 1 amino acid, approximately 500 amino acids, and approximately 400 amino acids. Acid, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 Amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 Amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately 20 amino acids, approximately 18 amino acids, approximately 16 Amino acids, approximately 14 amino acids, approximately 12 amino acids, approximately 10 amino acids, approximately 8 amino acids, approximately 6 amino acids No acids, between approximately 4 amino acids and approximately 2 amino acids; between approximately 2 amino acids and approximately 500 amino acids. Approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids Mino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids Mino acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately 20 amino acids, approximately 18 amino acids Mino acids, approximately 16 amino acids, approximately 14 amino acids, approximately 12 amino acids, approximately 10 amino acids, approximately 8 amino acids No acids, approximately 6 amino acids, or up to approximately 4 amino acids; from approximately 4 amino acids to approximately 500 amino acids. Approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids Mino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids Mino acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately 20 amino acids, approximately 18 amino acids Mino acids, approximately 16 amino acids, approximately 14 amino acids, approximately 12 amino acids, approximately 10 amino acids, approximately 8 amino acids No acids, or up to about 6 amino acids; from about 6 amino acids to about 500 amino acids, about 400 amino acids Acid, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 Amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 Amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately 20 amino acids, approximately 18 amino acids, approximately 16 Amino acids, approximately 14 amino acids, approximately 12 amino acids, approximately 10 amino acids, or approximately 8 amino acids Then; from approximately 8 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 20 0 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately 20 amino acids, approximately 18 amino acids, approximately 16 amino acids, approximately 14 amino acids, approximately Up to 12 amino acids, or approximately 10 amino acids; from approximately 10 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids 0.0 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids No acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately 20 amino acids, approximately 18 amino acids No acids, approximately 16 amino acids, approximately 14 amino acids, or up to approximately 12 amino acids; approximately 12 amino acids From approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 1 00 amino acids, approximately 90 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately Up to 20 amino acids, approximately 18 amino acids, approximately 16 amino acids, or approximately 14 amino acids; approximately 14 Approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids Acid, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids 0.0 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids No acids, approximately 20 amino acids, approximately 18 amino acids, or up to approximately 16 amino acids; approximately 16 amino acids From approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 1 00 amino acids, approximately 90 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids, approximately Up to 20 amino acids, or approximately 18 amino acids; from approximately 18 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids 0.0 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids No acids, approximately 35 amino acids, approximately 30 amino acids, approximately 25 amino acids, or up to approximately 20 amino acids. Approximately 20 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 20 0 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 amino acids, approximately 30 amino acids, also Or up to about 25 amino acids; from about 25 amino acids to about 500 amino acids, about 400 amino acids, Approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids 0.0 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, approximately 35 amino acids No acids, or up to about 30 amino acids; from about 30 amino acids to about 500 amino acids, about 400 amino acids Mino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, approximately 40 amino acids, also Or up to about 35 amino acids; from about 35 amino acids to about 500 amino acids, about 400 amino acids, Approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids No acids, approximately 70 amino acids, approximately 60 amino acids, approximately 50 amino acids, or up to approximately 40 amino acids. Approximately 40 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 20 0 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids, approximately 70 amino acids, approximately Up to 60 amino acids, or approximately 50 amino acids; from approximately 50 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids No acids, approximately 80 amino acids, approximately 70 amino acids, or up to approximately 60 amino acids; approximately 60 amino acids From approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, approximately 1 50 amino acids, approximately 100 amino acids, approximately 90 amino acids, approximately 80 amino acids, or approximately 70 amino acids Up to amino acids; approximately 70 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids Acid, approximately 200 amino acids, approximately 100 amino acids, approximately 90 amino acids, or approximately 80 amino acids Then; from approximately 80 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 2 From 00 amino acids, to approximately 100 amino acids, or up to approximately 90 amino acids; from approximately 90 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 300 amino acids, approximately 200 amino acids, or approximately Up to 100 amino acids; approximately 100 amino acids to approximately 500 amino acids, approximately 400 amino acids, approximately 3 00 amino acids, or up to approximately 200 amino acids; from approximately 200 amino acids to approximately 500 amino acids Approximately 400 amino acids, or up to approximately 300 amino acids; approximately 300 to approximately 500 amino acids up to 400 amino acids or approximately 400 amino acids; or from approximately 400 amino acids to approximately 500 amino acids It may have a length of .

[0254] CAR transmembrane domain The CARs provided herein also include transmembrane domains. In some embodiments, transmembrane domains are also included. The domains may originate from natural sources. In some embodiments, the transmembrane domains are optional. It may be derived from a membrane-bound or transmembrane protein. In the CARs described herein, Non-limited examples of transmembrane domains that can be used include the α, β, or ζ chains of T cell receptors, and C. D28, CD3ε, CD33, CD37, CD64, CD80, CD45, CD4, CD 5, CDS, CD9, CD16, CD22, CD86, CD134, CD137, or It may originate from CD154 (for example, at least its transmembrane sequence, or its transmembrane sequence (Including a portion). In one embodiment, the transmembrane domain is derived from the CD4 molecule. One embodiment Therefore, the transmembrane domain originates from the CD8 molecule.

[0255] In some embodiments, the transmembrane domain may be synthetic. For example, the transmembrane domain may be In some embodiments derived from synthetic sources, the transmembrane domain is a hydrophobic residue (e.g., ro It may contain (for example, primarily) isine and valine. The synthetic transmembrane domain has phenylalanine and tryptase at its terminus. A triplet of fan and varnish, at least one (for example, at least two, less It is thought to include at least three, at least four, at least five, or at least six. In some embodiments, the transmembrane domain of the CAR may include a CD8 hinge domain. ru.

[0256] In some embodiments, the transmembrane domain is naturally associated with the sequence within the cytoplasmic domain. In some embodiments, the transmembrane domain interacts with other members of the receptor complex. To minimize this, other transmembrane domains of that domain (e.g., same or different) One or more (e.g.,) to avoid binding to the transmembrane domain of surface membrane proteins. For example, two, three, four, five, six, seven, eight, nine, or ten amino acids It can be modified by exchange.

[0257] In some embodiments, the transmembrane domain of the CAR provided herein is CD3-ζ, Transmembrane suppositories for CD8, CD28, NKG2D, CD16, NKp44, or NKp46 Includes . In certain embodiments, the transmembrane domain of the CAR provided herein is C The transmembrane domains include D3-ζ, CD8, or CD28. In this department, the intracellular domains of CAR are as follows: CD27, CD28, 4-1BB, O From X40, CD30, CD40, PD-1, ICOS, and any combination thereof. It contains the co-stimulatory domain of a co-stimulatory molecule selected from the group.

[0258] CAR's intracellular (cytoplasmic) domain The intracellular domain is, for example, an immune effector cell, such as a T cell, an NK cell, or It is known to function in transmitting signals that trigger macrophage activation. It can be any polypeptide domain. Such a domain or motif is C The activation of T lymphocytes in response to the binding of the extracellular domain of AR to the target antigen is necessary. It can transmit primary antigen-binding signals. Examples of intracellular domains include ILR chains and C This includes, but is not limited to, D28, 4-1BB, and CD3ζ.

[0259] Typically, the intracellular domain is an ITAM (immune receptor tyrosine-based activation motif). ) includes.

[0260] In one embodiment, the intracellular domain is a CD3ζ signaling sequence (e.g., its ITA The M-containing portion is or contains the M-containing portion. In one embodiment, the intracellular domain is lymphatic It contains a globular receptor chain. In one embodiment, the intracellular domain is the TCR / CDR3 complex protein. It contains a substance. In one embodiment, the intracellular domain includes an Fc receptor subunit. In the application form, the intracellular domain includes the IL-2 receptor subunit.

[0261] The intracellular domains of CARs provided herein are two different classes of cytoplasmic signaling It can contain a signaling sequence that initiates antigen-dependent activation via the TCR. Signaling sequences (primary cytoplasmic signaling sequences) (e.g., CD3ζ cytoplasmic signaling sequences) , and co-stimulators that act in an antigen-independent manner to provide secondary or co-stimulatory signals One or more cytoplasmic sequences (secondary cytoplasmic signaling sequences) within a protein.

[0262] In a particular embodiment, the antigen-binding domain stimulates T cells when it binds to an antigen. A sufficient cytoplasmic arrangement of CD3ζ, and optionally, co-stimulation of T cells. One or more cytoplasmic sequences of the stimulant proteins (e.g., CD27, CD28, 4- 1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS , LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD2 44, CD80, LAG3, NKG2C, B7-H3, 2B4, DAP10, CD137 ligands that specifically bind to DAP12, CD83, and the ligands described herein Or one or more cytoplasmic arrangements of any of the ITAM sequences known in the art. A CAR comprising an intracellular signaling domain including a column is provided herein. In some embodiments, the entire intracellular signaling domain of the costimulatory protein is related to CAR It is included in the intracellular domain. In some embodiments, the intracellular domain is a co-stimulatory protein. Shortened portions of the intracellular signaling domains (e.g., CAR expression immunoeffectors) - Shortening of intracellular signaling domains that transmit effector function signals in cells. (Includes the portion that has been modified). Intracellular signaling domains that can be included in the intracellular domain. A non-limiting example of this is the initiation of signal transduction after antigen receptor engagement. The cytoplasmic arrangement of T cell receptors (TCRs) and co-receptors that work together, as well as the small At least one (for example, one, two, three, four, five, six, seven, eight, nine, and also Any of these sequences, including 10 substitutions, that have the same or nearly the same functional ability. Variants of this type can be listed.

[0263] In some embodiments, the intracellular domain of CAR contains the CD3ζ signaling domain, Any other desired cytoplasmic signaling pathway that is useful on its own or in the context of CAR It can be designed to include in combination with columns. In some embodiments, CAR cells The cytoplasmic domain may include the CD3ζ chain portion and the costimulatory cytoplasmic signaling sequence. Cytoplasmic signaling sequences are associated with co-stimulatory proteins (e.g., CD27, CD28, 4-IB). B(CD 137), OX40, CD30, CD40, PD-1, ICOS, Lymphocyte machine function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 CARs (which include cytoplasmic signaling sequences of ligands that specifically bind to CD83) It refers to a part of something.

[0264] In some embodiments, the cytoplasmic signaling sequence within the intracellular domain of CAR is Randa They are arranged in a specific order. In some embodiments, the cytoplasmic signals within the intracellular domain of the CAR The linkage sequence is linked to each other in a specific order. In some embodiments, a linker (for example) (or any of the linkers described herein) between different cytoplasmic signaling sequences It can be used to form a chain.

[0265] In some embodiments, the intracellular domain is associated with the cytoplasmic signaling sequence of CD3ζ and co Designed to include the cytoplasmic signaling sequence of the stimulating protein CD28. Morphologically, the intracellular domain consists of the cytoplasmic signaling sequence and co-stimulatory protein of CD3ζ. Designed to include the cytoplasmic signaling sequence of 4-IBB. In some embodiments, The intracellular domain consists of the cytoplasmic signaling sequence of CD3ζ and the co-stimulatory protein CD2 Designed to include cytoplasmic signaling sequences 8 and 4-1BB. Some embodiments Therefore, the intracellular domain does not contain the cytoplasmic signaling sequence 4-1BB.

[0266] In some embodiments, CAR is CD28, CD137 (also known as 4-1BB) ), CD134 (also known as OX40) and CD278 (also known as ICOS) It contains one or more co-stimulatory domains derived from proteins such as ( ). Morphologically, CAR does not contain a co-stimulatory domain derived from CD137.

[0267] In certain embodiments, the intracellular domain further includes cytokines. Morphologically, the intracellular domain is a self-cleaving domain (e.g., P2A self-cleaving peptide). ) and cytokines are further included, and cytokines are released by cleavage of the self-cleaving domain. In some embodiments, self-cleaving domains (e.g., P2A self-cleaving peptides) are produced. ) and cytokines are located at the C-terminus of CAR proteins and their intracellular domains. In some embodiments, the cytokines are: IL-12, IL-7, I L-13, IL-15, IL-4, IL-10, TNF-α, IFN-γ, TGF-β and One or more of the following: and CCL19. In one embodiment, the cytokine is IL- It is 12. In one embodiment, the cytokine is IL-7. In one embodiment, site The cytokine is IL-13. In one embodiment, the cytokine is IL-15. Morphologically, the cytokine is IL-4. In one embodiment, the cytokine is IL-10 In one embodiment, the cytokine is TNF-α. The cytokine is IFN-γ. In one embodiment, the cytokine is TGF-β. In this state, the cytokine is CCL19. The immunoglobulin is modified to express the cytokine. Epidemic effector cells are known in this art (e.g., Adachi et al, 2018, Nature). Biotechnology, doi:10.1038 / nbt.4086; Liu et al., 2019, J. Immunol., doi:10.4049 / jimmunol.1800033; Krenciute et al., 2017, Cancer Immunol. Res. 597):571-581, do i:10.1158 / 2326-6066,CIR-16-0376; see Liu et al., 2018, Leukemia 32:520-531). In certain embodiments, the immunotherapy described herein is used to express cytokines. Modification of effector cells was described in Adachi et al, 2018, Nature Biotechnology, doi:10.1038 / nbt.4086; Liu et al., 2019, J. Immunol., doi:10.4049 / jimmunol.1800033; et al., 2017, Cancer Immunol. Res. 597):571-581, doi:10.1158 / 2326-6066,CIR-16-0 376; or the same as described in Liu et al., 2018, Leukemia 32:520-531. Or, follow the method described there.

[0268] CAR-expressing immune effector cells In one embodiment, an immune system comprising any of the chimeric antigen receptors (CARs) described herein is present. Effector cells are provided herein. In certain embodiments, Transformation by nucleic acids encoding any of the chimeric antigen receptors (CARs) listed below. Immunoeffector cells are provided herein. In certain embodiments, Immunoeffects expressing any of the chimeric antigen receptors (CARs) described herein Tar cells are provided herein.

[0269] As immune effector cells that can be used to harbor or express CARs These include T cells, natural killer (NK) cells, and macrophages, but The definition is not limited to these. In one embodiment, immune effector cells are T cells (e.g., cell damage). (Harmful T cells). In one embodiment, the immune effector cells are NK cells. Morphologically, immune effector cells are macrophages.

[0270] In some embodiments, the immune effector cells provided herein are derived from peripheral blood, umbilical cord blood, and It is either isolated from the lymphatic fluid or amplified.

[0271] In some embodiments, the immune effector cells provided herein are (as described herein). (After the modification that causes the expression of CAR) it is autosomal to the subject to which it is administered. In certain embodiments, the immune effector cells provided herein are (in this specification (After the modification that causes the CAR described in the book to manifest) for the subject to which it is administered They are homogeneous. Allogeneic immune effector cells are used to create CAR-expressing immune effector cells. When prepared, immune effector cells that reduce the likelihood of graft-versus-host disease in the target population. You can choose to use immunoeffector cells with one or more immunosuppressants. They can be administered simultaneously. In some embodiments, immune effector cells are obtained from the subject. The polynucleotides expressing the CAR described herein are optionally increased. Therefore, it is transformed and further increased by choice.

[0272] In some embodiments, immune effector cells are involved in disease or condition (e.g., cancer, e.g., Derived from a patient with AML, and containing any of the antigens described herein (e.g., neoantigens). To express at least one CAR having specificity for the original (product) in vitro It is genetically modified. For example, antigens are presented by MHC class I proteins. Carcinoma neoantigens (e.g., MHC class I proteins, e.g., NPM1c:HLA-A) (Antigen containing a mutant nucleophosmin protein neoepitope that forms a complex with 2) This may be the case. In some of these embodiments, MHC class I proteins (e.g., NPM) are used. CARs that have specificity for cancer neoantigens presented by 1c:HLA-A2 Immune effector cells, genetically modified to express certain genes, are then used to treat cancer in patients. For example, it is administered to treat NPM1c-positive cancers (e.g., AML). In the application mode, immune effector cells are activated by the specific binding of ligands or antigens to CARs. This stimulates or induces, and is useful for treating the disease or condition of the same patient, at least It performs one effector function (e.g., cytokine induction). In some embodiments, it performs one effector function. Effector cells come into contact with or are in close proximity to the target or target cells (e.g., cancer cells). In some cases, it exerts its effector function (e.g., cytotoxic T cell response) on target cells. These are T cells (for example, cytotoxic T cells) (e.g., Chang and Chen (2017) Trends) See Mol Med 23(5):430-450).

[0273] Immunity involving CARs (for example, by the binding of the extracellular domain of CARs to cancer neoantigens) Stimulation of effector cells stimulates one or more anticancer activities of CAR immune effector cells. This can lead to the activation of CAR immunoeffector cells. For example, in some embodiments, this can result in the activation of CAR immunoeffector cells. Stimulation involves the cytolytic activity of CAR immune effector cells, including the secretion of cytokines or This can lead to an increase in helper activity.

[0274] In some embodiments, CAR effector cells (e.g., CAR T cells) are described in this specification. CARs that bind to any of the antigens listed in the book (e.g., NPM1c:HLA-A2) Includes molecules. In some embodiments, CAR molecules useful in the methods disclosed herein. Immune effector cells, including (for example, CAR T cells), are MHC class I proteins. NPM that has formed a complex with (or is presented by) HLA-A2 (for example) 1c neoepitope, e.g., NPM1c: contains an extracellular domain that binds to HLA-A2. It expresses CAR. In some embodiments, C is useful in the method disclosed herein. Immune effector cells containing AR molecules (e.g., CAR T cells) are NPM1c:HL It expresses a CAR containing an A-A2 binding domain.

[0275] Prolonged exposure of T cells to their cognitive antigens leads to depletion of effector function. This allows infected or transformed cells to persist. It induces immune checkpoint inhibition. Recently developed strategies for stimulating or activating host effector functions using drugs It has achieved success in treating several types of cancer. New evidence suggests that T cell depletion is the key to success. The importance of sustained long-lived antitumor activity by CAR T cells. This has been suggested to be a major obstacle. T cells collected from patients before CAR transduction. Differentiation state, and pre-transplant treatments the patient receives before reintroducing CAR T cells (e.g., A Adding or removing cytotoxic agents, fludarabine, or whole-body irradiation can lead to persistent CAR-T cell activity. This can significantly affect the cytotoxicity of the T cell population (anti-CD3 / CD28 or stimulated cells). (via) stimulation and (via cytokines, e.g., IL-2) increase in vit The culture conditions can also alter the differentiation state and effector function of CAR T cells (Gh oneim et al., (2016) Trends in Molecular Medicine 22(12):1000-1011).

[0276] Method for producing CAR-expressing immunoeffector cells An immune effector as described herein, comprising any of the CARs described herein. —Methods that can be used to generate any of the cells are provided herein. To be served.

[0277] In some embodiments, target immune effector cells (e.g., T cells) are treated with chimeric antigens. Receptor-mediated gene modification (Sadelain et al., Cancer Discov. 3:388-398, 2013) For example, prepare immune effector cells (e.g., T cells) and apply chimeric antigen receptors. Recombinant nucleic acids are introduced into patient-derived immune effector cells (e.g., T cells), and C AR cells are generated. In some embodiments, immune effector cells that do not originate from the target ( For example, T cells are genetically modified using chimeric antigen receptors. For example, some implementations In this scenario, immune effector cells (e.g., T cells) are used in "ready-made" adoptive cell therapy, for example. Universal chimeric antigen receptor T cells developed by Cellectis These are allogeneic cells that have been engineered to be used as UCART (Union Cell Arterial Arteries).

[0278] Using various different methods known in the art, the CAR described herein is coated Expression comprising any of the nucleic acids described herein, or nucleic acids encoding CARs as described herein. The vector can be introduced into immune effector cells (e.g., T cells). A non-limiting example of a method for introducing nucleic acids into effector cells (e.g., T cells) is: , lipofection, transfection (e.g., calcium phosphate transfection) Transfection using highly branched organic compounds, and the use of cationic polymers. Transfection using, dendrimer-based transfection, optical transfection Transfection, particle-based transfection (e.g., nanoparticle transfection) Transfers using a suction, or liposomes (e.g., cationic liposomes) injection, microinjection, electroporation, cell compression, sonopore Protoplast fusion, imparefection, hydrodynamic delivery, gene gun, Examples include gnetofection, viral transfection, and nucleofection. Furthermore, using the CRISPR / Cas9 genome editing technology known in this field... Then, CAR nucleic acids are introduced into immune effector cells (e.g., T cells), and / or other Inducing genetic modification (for example, as shown below) in immune effector cells (for example, T cells) It can enhance CAR T cell activi...

Claims

1. NPM that forms a complex with Class I major histocompatibility complex (MHC class I) proteins. An antibody that specifically binds to an antigen containing a 1c neoepitope, or an antigen-binding fragment thereof.

2. (a) the MHC class I protein alone, and / or (b) the MHC class I It does not bind to the control peptide that forms a complex with the protein, or it does not bind substantially. Optionally, the control peptide may be the NY-ESO-1 epitope or influenza The antibody according to claim 1, or its antigen-binding fragment, which is an Virus M1 epitope.

3. The aforementioned NPM1c neoepitope has amino acid sequence X １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ X ９ comprising, wherein X １ is selected from A, V, L or I, and X ２ is A, T, S, V, L, Select from I, M, or Q, X ３ is selected from Q or N, X ４ Choose from D or E Selected, X ５ is selected from L, I, V, M, A, or F, X ６ is from C, S, or A Selected, X ７ is selected from L, I, V, M, A, or F, X ８ is A, V, L or Selected from I, X ９ is selected from L, I, V, M or A as described in claim 1 or 2 The antibody, or its antigen-binding fragment.

4. X １ is selected from A or V, X ２ is selected from V, I, or L, X ３ But Q again X is selected from N, ４ If selected from D or E, X ５ is selected from L or I, X ６ is selected from C or S, X ７ is selected from V, L, or I, X ８ Is it A or V? Selected from, X ９ The antibody according to claim 3, wherein is selected from V, I, or L, or Antigen-binding fragment.

5. X １ A is X ２ is selected from V, I, or L, X ３ Q is X ４ is D Yes, X ５ L is X ６ C is X ７ L is X ８ A is X ９ V, An antibody according to claim 4, or an antigen-binding fragment thereof, selected from I or L.

6. The NPM1c neoepitope is AIQDLCLAV (SEQ ID NO: 1) or AIQD Any of claims 1 to 4, comprising an amino acid sequence selected from LCVAV (SEQ ID NO: 71) The antibody described in item 1, or its antigen-binding fragment.

7. The aforementioned NPM1c neoepitope is CLAVEEVSL (SEQ ID NO: 72), VEEVS LRK (SEQ ID NO: 73), AVEEVSLLR (SEQ ID NO: 74), AVEEVSLRK (distribution) (Column number 75), select the amino acid sequence from CLAVEEVSLRK (SEQ ID NO: 76) The antibody according to claim 1 or 2, or its antigen-binding fragment.

8. The neoepitope comprises the amino acid sequence AIQDLCLAV (SEQ ID NO: 1), An antibody or antigen-binding fragment thereof, as described in any one of items 1 to 6.

9. The neoepitope has a length of 7, 8, 9, 10, 11, or 12 amino acid residues. An antibody according to any one of claims 1 to 8, or an antigen-binding fragment thereof.

10. The aforementioned MHC class I protein is either an HLA-A*02 protein or HL The allele group A-A*02, as described in any one of claims 1 to 9. An antibody, or its antigen-binding fragment.

11. The aforementioned MHC class I protein is encoded by the HLA-A*02:01 allele. an antibody according to any one of claims 1 to 10, or an antigen-binding fragment thereof.

12. (i) Heavy chain variable region (VH) complementarity determination region (CDR) 1, VH CDR 2 and VH A VH including CDR3, wherein VH CDR1, VH CDR2 and VH CD R3 is the CDR of VH having the amino acid sequence of SEQ ID NO: 5, VH, and / or teeth (ii) Light chain variable region (VL) complementarity determination region (CDR) 1, VL CDR 2 and V A VL including L CDR3, wherein VL CDR1, VL CDR2 and VL C DR3 is a CDR of VL having the amino acid sequence of SEQ ID NO:

3. An antibody according to any one of claims 1 to 11, or an antigen-binding fragment thereof, comprising:

13. Heavy Chain Variable Region (VH) Complementarity Determination Region (CDR) 1, VH CDR 2, and VH CD VH containing R3, wherein the VH CDR1 has the amino acid sequence GTFFSSYA (Sequence ID) 9) has the amino acid sequence ISGSGGST (SEQ ID NO: 10) and the VH CDR2 has the amino acid sequence ISGSGGST Furthermore, the VH CDR3 has the amino acid sequence ARLGYPTTTLLPFDY (SEQ ID NO: 11 An antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, having the following characteristics:

14. Light chain variable region (VL) complementarity determination region (CDR) 1, VL CDR 2, and VL CD The VL further contains R3, and the VL CDR1 is an amino acid sequence QSISSY (sequence number) VL CD2 has the amino acid sequence AAS (SEQ ID NO: 7), and VL Claim 13, wherein CD3 has the amino acid sequence QQSYSPLT (SEQ ID NO: 8) An antibody, or its antigen-binding fragment.

15. It includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is the same as in Sequence ID No.

5. An amino acid sequence that is at least 90% identical to, or at least 95% identical to, an amino acid sequence It includes, and / or the VL is at least 90% identical to the amino acid sequence of SEQ ID NO:

3. Any of claims 1 to 14, comprising one or at least 95% identical amino acid sequences The antibody described in item 1, or its antigen-binding fragment.

16. It includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH is the A of Sequence ID No.

5. A claim comprising a amino acid sequence and / or the VL comprising the amino acid sequence of SEQ ID NO:

3. An antibody or antigen-binding fragment thereof, as described in any one of items 1 to 15.

17. The antibody is a human antibody, a humanized antibody, or a chimeric antibody, as described in any one of claims 1 to 16. An antibody, or its antigen-binding fragment.

18. Single lock Fv (scFv), Fv fragment, Fab fragment, Fab' fragment, F (ab') ２ fragment, Alternatively, the antibody according to any one of claims 1 to 17, which is a single-chain antibody molecule, or the antibody thereof. Original combined fragment.

19. The antibody according to claim 18, or its antigen-binding fragment, which is scFv.

20. The antibody according to claim 19, or its antigen binding cleavage, wherein the scFv is a human scFv. Piece.

21. The antibody or antigen according to claim 19 or 20, wherein the scFv comprises a linker. Combined fragment.

22. The antibody or antigen according to claim 21, wherein the linker is a peptide linker. Combined piece.

23. The antibody according to claim 22, wherein the peptide linker is a Gly-Ser linker. And that is the antigen-binding fragment.

24. The aforementioned Gly-Ser linker is (Gly4Ser) (Sequence No. 58), (Gly4S (Gly4 Ser) 2 (Sequence ID 59), (Gly4 Ser) 3 (Sequence ID 60), and (Gly4 The antibody according to claim 23, selected from the group consisting of Ser) 4 (Sequence ID 61), and It is the antigen-binding fragment.

25. The Gly-Ser linker performs the amino acid sequence SGSGGGSSG (SEQ ID NO: 4) The antibody according to claim 23, or its antigen-binding fragment.

26. The aforementioned scFv is at least 80% identical to the amino acid sequence of SEQ ID NO: 2, and at least 8 Having amino acid sequences that are 5% identical, at least 90% identical, or at least 95% identical. And, optionally, the scFv is (a) heavy chain variable region (VH) complementarity determination region (CDR ) 1. A VH including VH CDR2 and VH CDR3, wherein VH CDR1 is It has the amino acid sequence GFTFSSYA (SEQ ID NO: 9), and the VH CDR2 is an amino acid sequence The sequence ISGSGGST (SEQ ID NO: 10) is present, and the VH CDR3 is the amino acid sequence ARL VH having GYPTTTLLPFDY (Sequence ID 11); and / or (b) light Chain Variable Region (VL) Complementarity Determination Region (CDR) 1, VL CDR 2, and VL CDR 3 A VL containing, wherein the VL CDR1 has the amino acid sequence QSISSY (SEQ ID NO: 6) The VL CD2 has the amino acid sequence AAS (SEQ ID NO: 7), and the VL CD3 Claim 19 to An antibody or antigen-binding fragment thereof as described in any one of item 25.

27. Any one of claims 19 to 26, wherein the scFv has the amino acid sequence of SEQ ID NO:

2. The antibody described above, or its antigen-binding fragment.

28. An antibody, as described in any one of claims 1 to 17, or an antigen-binding fragment thereof.

29. The aforementioned antibodies are IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2 Selected from the group consisting of IgD and IgE antibody isotypes, as described in claim 28. An antibody, or its antigen-binding fragment.

30. Claim that the antibody is an IgG1 isotype or an IgG4 isotype. The antibody described in 29, or its antigen-binding fragment.

31. The antibody comprises the wild-type IgG1 heavy chain constant region or the wild-type IgG4 heavy chain constant region. The antibody according to claim 30, or its antigen-binding fragment.

32. The antibody comprises a mutant IgG1 heavy chain constant region or a mutant IgG4 heavy chain constant region. The antibody according to claim 30, or its antigen-binding fragment.

33. The antibody comprises a mutant IgG4 heavy chain constant region, and the mutant IgG4 heavy chain constant region is Substitutions: any of the following EU numbering schemes: S228P, L235E, or L235A The antibody according to claim 32, or its antigen-binding fragment, comprising one or a combination thereof. 。

34. Claims 28-30, 3, wherein the antibody comprises an Fc domain containing at least one mutation. An antibody or antigen-binding fragment thereof as described in any one of paragraphs 2 or 33.

35. The antibody according to any one of claims 1 to 34, wherein the antigen is present on the surface of cancer cells, It is the antigen-binding fragment.

36. The antibody according to claim 35, wherein the cancer is acute myeloid leukemia (AML), or the same. Antigen-binding fragment.

37. NPM that forms a complex with Class I major histocompatibility complex (MHC class I) proteins. Antigens containing 1c neoepitopes: 100 nM or less, 50 nM or less , 20 nM or less, 10 nM or less, 0.5 nM to 100 nM, Alternatively, the bonds are formed with an equilibrium dissociation constant (Kd) ranging from 1 nM to 15 nM, according to claims 1 to 36. Any of the antibodies described in item one, or their antigen-binding fragments.

38. A bispecific antibody that binds even more specifically to a second antigen on immune effector cells, The antigen-binding fragment is the antibody according to any one of claims 1 to 37, or the antibody thereof. Original combined fragment.

39. The effector cells are T cells, natural killer cells, or macrophages. The antibody according to claim 38, or its antigen-binding fragment.

40. The antibody or antigen according to claim 38 or 39, wherein the second antigen is CD3. Combined fragment.

41. The antibody according to claim 40, wherein the CD3 is human CD3 expressed on T cells. And that is the antigen-binding fragment.

42. The antibody according to claim 38 or 39, wherein the second antigen is NKp46, or the Antigen-binding fragment.

43. Claim 42 states that the NKp46 is human NKp46 expressed on NK cells. An antibody, or its antigen-binding fragment.

44. The antibody according to claim 38 or 39, wherein the second antigen is CD16A, or the antibody or the Antigen-binding fragment.

45. Claim 44 states that the CD16A is human CD16A expressed on NK cells. An antibody, or its antigen-binding fragment.

46. The second antigens mentioned above are CD40, CD47, 4-1BB, TGF-β, LAG-3, and PD. -1, TIM-3, CTLA-4, OX40, NKp30, NKG2A, NKG2D, also The antibody or antigen-binding fragment thereof according to claim 38 or 39, wherein is DNAM-1.

47. A purified antibody according to any one of claims 1 to 46, or its antigen binding cleavage. Piece.

48. An isolated nucleic acid, which is an antibody according to any one of claims 1 to 46, or an antibody thereof. A nucleic acid containing a nucleic acid sequence that encodes the original binding fragment.

49. The isolated nucleic acid according to claim 48, comprising the nucleotide sequence of sequence number 12.

50. An expression vector comprising the nucleic acid according to claim 48 or 49.

51. Cells transformed with the expression vector described in claim 50.

52. To produce an antibody or antigen-binding fragment thereof according to any one of claims 1 to 46 A method comprising expressing the cells described in claim 51, the antibody or its antigen-binding fragment. A method including the step of maintaining conditions that enable this.

53. Claim 52 further comprises the step of purifying the antibody or its antigen-binding fragment. Method of loading.

54. A therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 47 A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

55. Chimeric antigen receptors including intracellular domain, transmembrane domain and extracellular binding domain ( A CAR polypeptide wherein the extracellular binding domain is a Class I major histocompatibility complex Anti- A CAR polypeptide that specifically binds to the source.

56. Chimeric antigen receptors including intracellular domain, transmembrane domain and extracellular binding domain ( A CAR polypeptide, wherein the extracellular binding domain is as described in claims 1 to 27 and 38 A CAR polypeptide containing an antibody or antigen-binding fragment thereof, as described in any one of items ~46. Do.

57. The aforementioned transmembrane domains are CD3-zeta, CD8, CD28, NKG2D, CD16, C according to claim 55 or 56, comprising a transmembrane domain of NKp44 or NKp46. AR polypeptide.

58. The intracellular domains are CD27, CD28, 4-1BB, OX40, CD30, CD It consists of 40, PD-1, ICOS, 2B4, DAP10, CD137, and DAP12. Includes one or more co-stimulatory domains of one or more co-stimulatory molecules selected from the group. or the CAR polypeptide according to any one of claims 55 to 57.

59. The intracellular domain is the CD3-zeta signaling domain and the 4-1BB costimulator The main component includes the transmembrane domain which includes the CD8 transmembrane domain, and the CAR polyp The CAR according to any one of claims 55 to 58, wherein the Chid further includes a CD8 hinge region. Polypeptide.

60. The intracellular domain contains the amino acid sequence shown in Sequence ID No. 27, which is a CD3-zetase The 4-1BB synovial, which includes the Gunal signaling domain and the amino acid sequence shown in SEQ ID NO: 26, The CAR polypeptide contains a CD8 transmembrane domain and a CD8 hinge. The region includes the CD8 transmembrane domain and the CD8 hinge region as shown in Sequence ID No.

25. The amino acid sequence comprises the extracellular binding domain, and the extracellular binding domain breaks the binding of the antibody or its antigen. Claim comprising a piece and a leading sequence including the amino acid sequence shown in SEQ ID NO: 23 A CAR polypeptide as described in any one of items 55 to 59.

61. The antibody, or its antigen-binding fragment, in the extracellular binding domain, SEQ ID NO: 2 The amino acid sequence shown in 4, or the amino acid sequence of SEQ ID NO: 24, is at least 70% identical. Well, at least 75% identical, at least 80% identical, at least 85% identical, at least scFv containing amino acid sequences that are at least 90% identical, or at least 95% identical. The CAR polypeptide according to any one of claims 55 to 60.

62. The intracellular domain further comprises a self-cleaving peptide sequence and a cytokine, The cytokine is released by the cleavage of the self-cleaving peptide, according to any of claims 55 to 61. A CAR polypeptide as described in any one of the items.

63. The cytokines mentioned above are IL-12, IL-7, IL-13, IL-15, TNF-α, The CAR polypeptide according to claim 62, which is IFN-γ or CCL19.

64. The amino acid sequence shown in SEQ ID NO: 22, or at least the amino acid sequence of SEQ ID NO: 22 70% identical, at least 75% identical, at least 80% identical, at least 85% Contains identical, at least 90% identical, or at least 95% identical amino acid sequences or the CAR polypeptide according to claim 55 or 56.

65. An isolated CAR polypeptide encoding the CAR polypeptide according to any one of claims 55 to 64 nucleic acid.

66. The nucleotide sequence of SEQ ID NO: 30, or the nucleotide sequence of SEQ ID NO: 30 and at least 70% identical, at least 75% identical, at least 80% identical, at least 85% Identical, at least 90% identical, or at least 95% identical nucleotide sequences Including the isolated nucleic acid according to claim 65.

67. An expression vector comprising an isolated nucleic acid according to claim 65 or 66, wherein the virus An expression vector that is either an expression vector or a nonviral expression vector.

68. The expression vector is a viral expression vector, and the viral expression vector is a wrench The expression vector according to claim 67, which is a viral expression vector.

69. Cells transformed by the expression vector according to claim 67 or 68.

70. A cell expressing the CAR polypeptide according to any one of claims 55 to 64.

71. The cells are immune effector cells, and the expression of the CAR polypeptide is the immune Effector cells are combined with Class I major histocompatibility complex (MHC Class I) proteins. Targeting cancer cells that express antigens containing the formed NPM1c neoepitope, The cell according to claim 69 or 70.

72. The aforementioned MHC class I protein is either an HLA-A*02 protein or HL The cell according to claim 71, encoded by the A-A*02 allele group.

73. The aforementioned immune effector cells substantially target cancer cells expressing wild-type NPM1. The cells according to claim 71 or 72, which induce the killing of the cells thereafter.

74. The cell according to any one of claims 69 to 73, wherein the cell is a T cell.

75. The aforementioned T cells are human CD8 ＋ The cell according to claim 74, which is a T cell.

76. The cell is a natural killer (NK) cell, according to any one of claims 69 to 73. The cells described.

77. The cell according to any one of claims 69 to 73, wherein the cell is a macrophage.

78. Any of claims 71 to 77, wherein the cancer cells are acute myeloid leukemia (AML) cells. The cells described in item 1.

79. A medical device comprising the cells according to any one of claims 69 to 78 and a pharmaceutically acceptable carrier. A pharmaceutical composition.

80. A method for producing cells according to any one of claims 69 to 78, (i) A step of purifying cells from target peripheral blood mononuclear cells (PMBCs), (ii) Optionally, the cells are subjected to an anti-CD3 antibody or its antigen-binding fragment and / or Alternatively, the step involves activation by an anti-CD28 antibody or its antigen-binding fragment. (iii) Transduction into the cells by the expression vector according to claim 67 or 68 Steps (iv) the step of isolating the cells expressing the CAR polypeptide, and (v) Optionally, a step of increasing the size of the isolated cells. A method that includes this.

81. A method for producing cells according to any one of claims 69 to 78, (i) Induce pluripotent stem cells (iPSCs) and differentiate them into immune effector cells. Top, (ii) The immunoeffector molecules expressed by the expression vector according to claim 67 or 68 Steps to introduce transduction into cells, (iii) Isolating the immune effector cells expressing the CAR polypeptide. Step, and (iv) Optionally, a step of increasing the size of the isolated immune effector cells. A method that includes this.

82. The method according to claim 81, wherein the immune effector cells are NK cells.

83. The method according to claim 81, wherein the immune effector cell is a macrophage.

84. A method for treating cancer in a person requiring cancer treatment, the method including the cancer The cell surface of the cell forms a complex with major histocompatibility complex (MHC class I) proteins. The formed NPM1c neoepitope is presented, and the method is any one of claims 1 to 47. The antibody described in the item, or its antigen-binding fragment, or the antibody described in any one of claims 69 to 78. Cells, or the pharmaceutical composition according to claim 54 or 79, to treat the cancer. A method comprising the step of administering a certain amount to the subject.

85. The method according to claim 84, wherein the cancer is acute myeloid leukemia (AML).

86. The method for treating cancer is a method for reducing the amount of cancer, or a method for survival in the subject. The method according to claim 84 or 85, which is a method for extending the period.

87. In patients requiring treatment for acute myeloid leukemia (AML), a method for treating AML. The antibody described in any one of claims 1 to 47, or the antigen-binding fragment thereof, claim The cells described in any one of items 69 to 78, or the pharmaceutical product described in claim 54 or 79. A method comprising the step of administering a composition to a subject in an amount sufficient to treat the AML. 。

88. Any of claims 85 to 87, wherein the AML is recurrent AML or refractory AML. The method described in item 1.

89. A method for preventing relapse of AML in a subject who is in remission from AML, according to claim 1 An antibody according to any one of claims 47, or an antigen-binding fragment thereof, according to claims 69 to 78. The cells described in either one of the claims, or the pharmaceutical composition described in claim 54 or 79, A method including the step of administering to an elephant.

90. Before the administration step, whether or not the subject expresses NPM1c, or the subject The system detects whether or not the subject has an NPM1c mutation in the NPM1 gene, and the subject is NPM1 If c is expressed or the NPM1c mutation is present, proceed to the administration step. The method according to any one of claims 84 to 89, including the method described in any one of claims 84 to 89.

91. Any of claims 84 to 90, wherein the administration is intravenous, intrathecal, intraosseous, or intraspinal. The method described in item 1.

92. Claim 8 further comprises the step of administering one or more additional therapeutic agents or procedures. The method described in any one of items 4 to 91.

93. The aforementioned additional therapeutic agents are inhibitors of immune checkpoint molecules, and optionally, the aforementioned immune The disease checkpoint molecule is TIM-3, PD-1, PD-L1, or CTLA-4. The method according to claim 92, wherein the inhibitor is optionally an antibody.

94. In the manufacture of a pharmaceutical product for treating cancer in a target, any one of claims 1 to 47 The antibody described in the item, or its antigen-binding fragment, or the antibody described in any one of claims 55 to 64. CAR polypeptide, cells according to any one of claims 69 to 78, or claim 54 Alternatively, the use of the pharmaceutical composition described in 79, wherein the cell surface of the cancer-containing cell is NPM1c complexed with major histocompatibility complex I (MHC class I) protein. Present a neoepitope; optionally, the use of such neoepitope may be one or more additional therapeutic agents or This is a combination of procedure and usage.

95. The method according to any one of claims 84 to 93, wherein the subject is a human, or Use as described in section 94.

96. (i) The antibody according to any one of claims 1 to 47, or the antigen-binding fragment thereof, claim A CAR polypeptide according to any one of claims 55 to 64, or any one of claims 69 to 78. Cells as described in paragraph 1, or the pharmaceutical composition as described in claim 54 or 79; (ii) optional (iii) Selective use of one or more additional therapeutic agents, and for the treatment of cancer in the target. A kit including one or more containers with instructions for use.

Images