Proteins containing the Kallikrein-related Peptidase 2 antigen-binding domain and their use

Abstract

To provide a next-generation kallikrein-related peptidase 2 (hK2) binding domain for therapeutic and diagnostic purposes. [Solution] Embodiments of the present invention provide isolated proteins comprising an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2), including monospecific and bispecific antibodies. Additional embodiments of the present invention provide polynucleotides encoding hK2-specific proteins, vectors, host cells, and methods for producing and using them.

Description

[Technical Field]

[0001] (Sequence Listing) This application has been submitted electronically in ASCII format, and the entire application is by reference. This specification includes sequence listings. The above ASCII copy is dated June 2, 2020. Created on the 3rd, named JBI6125USNP1_SL.txt, size 76 It is 4,265 bytes.

[0002] (Field of Invention) The present invention relates to an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2). The antigen-binding domain that binds to the hK2 protein, and the polynucleo that encodes it. The present invention provides cytoplasm, vectors, host cells, and methods for producing and using them.

[0003] (background) Prostate cancer is the second most common cancer and the sixth leading cause of cancer death in men. 14% (903,500) of all new cancer cases and 6% of all cancer deaths in men worldwide (258,400) is accounted for. The process from diagnosis to death of prostate cancer is influenced by the severity of the disease, hormones Mon's condition and presence or absence of detectable metastases: localized disease, detectable after radiation therapy or surgery The presence of elevated prostate-specific antigen (PSA) levels without metastasis, and the non-castrated stage or Based on clinical metastasis during the castration phase, it is best classified as a series of clinical stages. Surgery, radiation therapy, or a combination of both can be curative for patients with localized diseases. It is possible, but a significant proportion of these patients do not use elevated PSA levels as evidence. The disease is recurrent, and this is particularly true in high-risk groups, where metastasis occurs, i.e., the end of the disease. This can sometimes lead to a transition to the terminal stage of the disease.

[0004] Androgen depletion therapy (ADT) is the standard treatment, and the generally predictable outcome is P A decrease in SA, a stable phase in which the tumor does not grow, followed by an increase in PSA, and then castration-resistant disease. It is a regrowth. For many years, ADT has been the standard treatment for patients with metastatic prostate cancer. there were.

[0005] Kallikrein-related peptidase 2 (hK2, HK2) is the androgen receptor (AR). It is a trypsin-like enzyme that is driven by and specifically expressed in prostate tissue and prostate cancer. 2 is activated by the transmembrane protease, serine 2 (TMPRSS2), and in the prostate It is secreted into the ducts, where it cleaves semenogerin, an extracellular matrix in the ejaculate fluid, and then the sperm It initiates a cascade that enhances the motility of the cells. hK2 expression is present in prostate and prostate cancer tissue. Although limited, breast cancer after the AR pathway has been appropriately activated by steroid hormones has recently been observed. It has been proven that hK2 is detectable in strains and primary patient samples (U.S. Patent Application Publication). (Case No. 2018 / 0326102). Similar to PSA, when the prostate gland is enlarged or undergoes malignant transformation... When highly structured organization is impaired, catalytically inactive hK2 can be released into the blood. Sexual release occurs.

[0006] Next-generation hK2-binding domains are needed for therapeutic and diagnostic purposes.

[0007] (overview) This disclosure relates to an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2). The isolated protein contains an antigen-binding domain that binds to hK2, which includes the following: Provides isolated proteins: Heavy chain complementarity determination region (HCDR) 1 of the heavy chain variable region (VH) of sequence number 137, HCD Determination of light chain complementarity of R2, HCDR3, and the light chain variable region (VL) of Sequence ID No. 138 Regions (LCDR)1, LCDR2, and LCDR3; or HCD of VH of sequence number 137 R1, HCDR2, and HCDR3, and LCDR1 of VL of sequence number 138, LCD R2 and LCDR3; or HCDR1, HCDR2, and HC of VH of Sequence ID No. 162 DR3, and LCDR1, LCDR2, and LCDR3 of VL of sequence number 163; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and sequence number 16 LCDR1, LCDR2, and LCDR3 of VL 5; or HC of VH of SEQ ID NO: 166 DR1, HCDR2, and HCDR3, as well as LCDR1 and LCDR1 of the VL of sequence number 167. DR2 and LCDR3; or HCDR1, HCDR2, and H of VH in sequence number 168 CDR3, and LCDR1, LCDR2, and LCDR3 of VL of sequence number 169; also This refers to HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and sequence number 2 LCDR1, LCDR2, and LCDR3 of the 05 VL.

[0008] This disclosure also relates to isolated antigen-bound drugs that bind to kallikrein-related peptidase 2 (hK2). The main antigen-binding domain that binds to hK2 is the heavy chain variable region of SEQ ID NO: 75. The isolated antigen-binding domain includes the VH region and the VL light chain variable region of SEQ ID NO: 74. To provide.

[0009] This disclosure also relates to the isolation antigen binding of hK2 containing specific VH and VL amino acid sequences. We provide domains.

[0010] This disclosure also relates to antigen-binding domains that bind to kallikrein-related peptidase 2 (hK2). An isolated multispecific protein containing an antigen-binding domain that binds to hK2 This provides isolated multispecific proteins, including: the heavy chain variable region of SEQ ID NO: 137 ( The heavy chain complementarity-determining regions (HCDRs) 1, HCDR2, and HCDR3 of VH, and the sequence Light chain complementarity determination regions (LCDRs) 1 and 2 of the light chain variable region (VL) number 138. and LCDR3; or HCDR1, HCDR2, and HCDR3 of VH of Sequence ID No. 137 , and LCDR1, LCDR2, and LCDR3 of VL of sequence number 138; or sequence number HCDR1, HCDR2, and HCDR3 of sequence number 162, and V of sequence number 163 LCDR1, LCDR2, and LCDR3 of L; or HCDR1 of VH of sequence number 164 , HCDR2, and HCDR3, as well as LCDR1, LCDR2 of the VL of Sequence ID No. 165 , and LCDR3; or HCDR1, HCDR2, and HCDR of VH of Sequence ID No. 166 3, and LCDR1, LCDR2, and LCDR3 of VL of sequence number 167; or sequence VH HCDR1, HCDR2, and HCDR3 of number 168, and sequence number 169 LCDR1, LCDR2, and LCDR3 of VL; or HCDR of VH of SEQ ID NO: 204 1, HCDR2, and HCDR3, and LCDR1, LCDR1 of VL of Sequence ID No. 205 2, and LCDR3. In certain embodiments, the Disclosure relates to kallikrein-related peptidase 2. An isolated multispecific protein containing an antigen-binding domain that binds to (hK2), The antigen-binding domain that binds to hK2 is the heavy chain phase of the heavy chain variable region (VH) of Sequence ID No. 162. Complementary determination regions (HCDR) 1, HCDR2, and HCDR3, and the mild of sequence number 163 Light chain complementarity determination regions (LCDRs) 1, 2, and 3 of the chain variable region (VL) This provides isolated, multispecific proteins, including [specific protein name].

[0011] This disclosure also relates to antigen-binding domains that bind to kallikrein-related peptidase 2 (hK2). An isolated multispecific protein containing an antigen-binding domain that binds to hK2 , including the heavy chain variable region (VH) of SEQ ID NO: 75 and the light chain variable region (VL) of SEQ ID NO: 74 , provides isolated multispecific proteins.

[0012] This disclosure also relates to antigen-binding domains that bind to hK2, including specific VH and VL amino acid sequences. This provides isolated, multispecific proteins containing yin.

[0013] This disclosure also relates to antigen-binding domains that bind to kallikrein-related peptidase 2 (hK2). This provides isolated chimeric antigen receptors (CARs) containing [a specific component].

[0014] This disclosure also relates to antigen-binding domains that bind to kallikrein-related peptidase 2 (hK2). An isolated chimeric antigen receptor (CAR) containing hK2, wherein the antigen-binding domain that binds to hK2 The company provides isolated chimeric antigen receptors (CARs), including the following: the weight of SEQ ID NO: 137 Heavy chain complementarity determination regions (HCDRs) 1, HCDR2, and HCDR3 of the chain variable region (VH) , and the light chain complementarity determination region (LCDR) 1 of the light chain variable region (VL) of sequence number 138, LCDR2 and LCDR3; or HCDR1, HCDR2, and VH of Sequence ID No. 137 HCDR3, and LCDR1, LCDR2, and LCDR3 of the VL of sequence number 138 ; or HCDR1, HCDR2, and HCDR3 of VH of sequence number 162, and sequence number LCDR1, LCDR2, and LCDR3 of VL of number 163; or VH of sequence number 164 HCDR1, HCDR2, and HCDR3, and VL of sequence number 165, LCDR1 ,LCDR2, andLCDR3; or HCDR1, HCDR2 of VH of sequence number 166, and HCDR3, and LCDR1, LCDR2, and LCDR of VL of Sequence ID No. 167 3; or HCDR1, HCDR2, and HCDR3 of VH of sequence number 168, and sequence LCDR1, LCDR2, and LCDR3 of VL number 169; or V of sequence number 204 H's HCDR1, HCDR2, and HCDR3, and VL's LCDR of sequence number 205 1, LCDR2, and LCDR3. In certain embodiments, this disclosure relates to kallikrein-related particles. Isolated chimeric antigen receptor (CA) containing an antigen-binding domain that binds to plutidase 2 (hK2) R) wherein the antigen-binding domain that binds to hK2 is the heavy chain variable region of Sequence ID No. 162. Heavy chain complementarity determination regions (HCDRs) 1, HCDR2, and HCDR3 of the region (VH), and Light chain complementarity determination region (LCDR) 1 of the light chain variable region (VL) of sequence number 163, LCDR The present invention provides isolated chimeric antigen receptors (CARs), including 2 and LCDR3.

[0015] This disclosure also relates to antigen-binding domains that bind to kallikrein-related peptidase 2 (hK2). An isolated chimeric antigen receptor (CAR) containing hK2, wherein the antigen-binding domain that binds to hK2 The 'IN' refers to the heavy chain variable region (VH) of sequence number 75 and the light chain variable region (VL) of sequence number 74. This provides isolated chimeric antigen receptors (CARs) that include [the specified element].

[0016] This disclosure also relates to a first antigen-binding domain that binds to hK2 and a lymphocyte antigen (CD3) This provides an isolated, multispecific protein containing a second antigen-binding domain that binds to ( ).

[0017] In certain embodiments, the disclosure relates to a first antigen-binding domain that binds to hK2 and lymph An isolated multispecific protein containing a second antigen-binding domain that binds to a globular antigen, This provides an isolated, multispecific protein that is an anti-hK2 / anti-CD3 protein.

[0018] This disclosure also includes an immunoconjugate comprising an isolated antigen-binding domain that binds to hK2 of this disclosure. We provide the gate.

[0019] This disclosure also includes isolated proteins containing an antigen-binding domain that binds to hK2 of this disclosure. It provides immune conjugates.

[0020] This disclosure also relates to an isolated, multispecific antigen containing an antigen-binding domain that binds to hK2 of this disclosure. It provides immunoconjugates containing proteins.

[0021] This disclosure also includes an isolated CAR comprising an antigen-binding domain that binds to hK2 of this disclosure. We provide immune conjugates.

[0022] This disclosure also relates to a pharmaceutical composition comprising an isolated antigen-binding domain that binds to hK2 of this disclosure. provide.

[0023] This disclosure also includes isolated proteins containing an antigen-binding domain that binds to hK2 of this disclosure. The invention provides a pharmaceutical composition.

[0024] This disclosure also relates to an isolated, multispecific antigen containing an antigen-binding domain that binds to hK2 of this disclosure. The present invention provides a pharmaceutical composition containing protein.

[0025] This disclosure also provides pharmaceutical compositions comprising the isolated multispecific proteins of this disclosure.

[0026] This disclosure also includes an isolated CAR comprising an antigen-binding domain that binds to hK2 of this disclosure. To provide a pharmaceutical composition.

[0027] This disclosure also encodes an isolated antigen-binding domain that binds to hK2 of this disclosure. Provides free polynucleotides.

[0028] This disclosure also provides isolated proteins containing an antigen-binding domain that binds to hK2. This provides isolated polynucleotides.

[0029] This disclosure also relates to an isolated, multispecific antigen containing an antigen-binding domain that binds to hK2 of this disclosure. We provide isolated polynucleotides that encode proteins.

[0030] This disclosure also encodes an isolated CAR containing an antigen-binding domain that binds to hK2 of this disclosure. This provides isolated polynucleotides.

[0031] This disclosure also provides vectors comprising the polynucleotides of this disclosure.

[0032] Furthermore, the present invention provides a host cell comprising the polynucleotide or vector of this disclosure. .

[0033] This disclosure also relates to a method for treating hK2-expressing cancer in a subject, wherein a therapeutically effective amount of h A protein containing an antigen-binding domain that binds to K2, and the antigen-binding domain that binds to hK2. A protein containing an antigen-binding domain that binds to the hK2, and the hK2 A CAR-T cell containing an antigen-binding domain that binds to the immunoconjugate of this disclosure, or the present invention The indicated pharmaceutical composition is administered for a sufficient amount of time to treat the hK2-expressing cancer. The present invention provides a method that includes administering the substance to a target subject.

[0034] This disclosure also relates to a method for reducing the amount of hK2-expressing tumor cells in a subject, wherein hK2 A protein containing an antigen-binding domain that binds to hK2, and an antigen-binding domain that binds to the hK2. a multispecific protein containing an antigen-binding domain that binds to the hK2, and a protein that binds to the hK2 A CAR-T containing an antigen-binding domain, an immune conjugate of the present disclosure, or the present disclosure The pharmaceutical composition is administered over a period of time sufficient to reduce the amount of hK2-expressing tumor cells. The present invention provides a method, including administration to elephants.

[0035] This disclosure also relates to a method for preventing the engraftment of hK2-expressing cancer in a subject, wherein hK2 is linked to A protein containing an antigen-binding domain that binds to the hK2, A multispecific protein containing an antigen-binding domain that binds to the hK2, which binds to the hK2 CAR-T receptors containing an antigen-binding domain, the immune conjugates of this disclosure, or pharmaceuticals of this disclosure The composition is administered to a subject in need to establish hK2-expressing cancer in that subject. To provide methods, including those for preventing [the disease].

[0036] This disclosure also relates to non-cancerous conditions in subjects at risk of developing an hK2-expressing oncological condition. A method for treating the condition comprising an antigen-binding domain that binds to hK2, and an anti- A protein containing a protobinding domain, and a multi-functional protein containing an antigen-binding domain that binds to the hK2. isomeric proteins, CAR-T proteins containing an antigen-binding domain that binds to the hK2, and the exemption of this disclosure By administering the epidemic conjugate, or the pharmaceutical composition of this disclosure, to a subject in need thereof, The present invention provides a method for treating the non-cancerous condition.

[0037] This disclosure also relates to a method for treating prostate cancer in a subject, comprising a therapeutically effective dose of hK2 A protein containing an antigen-binding domain that binds to hK2, and an antigen-binding domain that binds to the hK2. a multispecific protein containing an antigen-binding domain that binds to the hK2, and a protein that binds to the hK2 A CAR-T containing an antigen-binding domain, an immune conjugate of the present disclosure, or the present disclosure The pharmaceutical composition is used for a sufficient amount of time to treat the prostate cancer as needed. The present invention provides a method that includes administering the substance to a target.

[0038] This disclosure also relates to a method for treating breast cancer in a subject, comprising a therapeutically effective dose of hK2. A protein containing an antigen-binding domain that binds to the hK2, A multispecific protein containing an antigen-binding domain that binds to the hK2, which binds to the hK2 CAR-T receptors containing an antigen-binding domain, the immune conjugates of this disclosure, or pharmaceuticals of this disclosure The composition is administered to a subject in need for a sufficient amount of time to treat the breast cancer. The present invention provides a method that includes administering a substance.

[0039] This disclosure also relates to a method for detecting prostate cancer or breast cancer in a subject, and the immunotherapy described herein. The conjugate is administered to the subject, and the immune conjugate is bound to hK2. The present invention provides a method for detecting prostate cancer or breast cancer, including detecting a certain substance. .

[0040] This disclosure also relates to an antigen-binding domain that binds to hK2, and an antigen-binding domain that binds to said hK2. The protein containing the main component, and the multispecific protein containing the antigen-binding domain that binds to the hK2. Protein, CAR-T containing an antigen-binding domain that binds to hK2, immunocondylosis of the present disclosure A kit is provided which includes a cegate or a pharmaceutical composition of the present disclosure.

[0041] This disclosure also relates to the anti-idiota that binds to the antigen-binding domain of hK2 in this disclosure. We provide IP antibodies.

[0042] This disclosure also includes an extracellular domain containing an antigen-binding domain that binds to hK2, and a transmembrane domain. The main and an intracellular signaling domain optionally containing at least one co-stimulatory domain. The present invention provides a chimeric antigen receptor (CAR) containing n and . [Brief explanation of the drawing]

[0043] The foregoing will become clear from a more detailed description of the exemplary embodiments shown in the accompanying drawings below. It will likely happen. TM: Transmembrane. [Figure 1] The sequence alignments of the VH domains of mu11B6 (sequence number 125), hu11B6 (sequence number 5), KL2B357 (sequence number 159), KL2B358 (sequence number 161), KL2B359 (sequence number 139), KL2B360 (sequence number 159), HCF3 (sequence number 6), and HCG5 (sequence number 4) are shown. [Figure 2] The sequence alignments of the VL domains of mu11B6 (sequence number 124), hu11B6 (sequence number 2), KL2B357 (sequence number 160), KL2B358 (sequence number 140), KL2B359 (sequence number 140), KL2B360 (sequence number 140), LDC6 (sequence number 1), and LCB7 (sequence number 3) are shown. [Figure 3] This figure shows the binding epitopes of selected hK2 antibodies mapped onto the hK2 antigen sequence. In this figure, each sequence is disclosed as sequence number 467. [Figure 4A] This shows the binding of hybridoma supernatant to primary human T cells. Clonal UCHT1 was used as a positive control (Figure 1B), and the mouse IgG1 isotype (mIgG1) was used as a negative control. [Figure 4B] This shows the binding of hybridoma supernatant to primary human T cells. Clonal UCHT1 was used as a positive control (Figure 1B), and the mouse IgG1 isotype (mIgG1) was used as a negative control. [Figure 5] This shows the binding of the anti-CD3 scFv variant expressed in Escherichia coli (E. coli) to CD3. [Figure 6] The alignment of the VL regions of CD3B815 (sequence number 249), CD3W244 (sequence number 250), CD3W245 (sequence number 251), CD3W246 (sequence number 252), CD3W247 (sequence number 253), and CD3W248 (sequence number 254) is shown. [Figure 7] The graph shows the results of hydrogen-deuterium exchange mass spectrometry (HDX-MS) of CD3W245 (CD3ε:CD3W245) bound to human CD3ε and OKT3 (CD3ε:OKT) bound to CD3ε. The amino acid sequences shown represent residues 2-93 of the 105 residues in the ECD domain of CD3ε, corresponding to residues 1-91 of CD3ε SEQ ID NO: 340. A single underline indicates a segment in which the deuteration level was reduced by 10%-30% in the presence of the antibody compared to CD3ε alone, and a double underline indicates a segment in which the deuteration level was reduced by >30%. [Figure 8A] This shows the in vitro target cell cytotoxicity of the KL2B×CD3 bispecific molecule, measured in real time by the incuCyte imaging system, for quantifying target cell death. [Figure 8B] This shows the in vitro target cytotoxicity of the KL2B×CD3 bispecific molecule, as measured by the fluorescent caspase 3 / 7 reagent for measuring apoptosis signals from target cell death. [Figure 9A] This demonstrates in vitro T cell activation and proliferation by a KLK2 × CD3 bispecific antibody, by showing the frequency of CD25-positive cells at different doses. [Figure 9B] This demonstrates in vitro T cell activation and proliferation by a KLK2 × CD3 bispecific antibody by showing the frequency of cells entering the proliferation gate. [Figure 10A] This demonstrates in vitro T-cell INF-γ release induced by a KLK2 x CD3 bispecific antibody. [Figure 10B] This demonstrates in vitro T-cell TNF-α release induced by a KLK2 x CD3 bispecific antibody. [Figure 11] The blueprint for the hK2-binding chimeric artificial receptor (CAR) is shown. hK2-binding scFv was cloned into various CARs in either VH-VL or VL-VH orientation. [Figure 12A] This shows hK2 CAR expression on the surface of T cells. Primary human T cells were electroporated with either no mRNA (mock), or 10 μg of mRNA expressing either the hK2 scFv CAR or an unrelated control CAR. 24 hours after electroporation, CAR surface expression was measured by flow cytometry after staining with PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (upper figure), or PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (lower figure), or PE conjugated with biotinylated hK2 (1 μg / mL) and streptavidin. [Figure 12B] This shows hK2 CAR expression on the surface of T cells. Primary human T cells were electroporated with either no mRNA (mock), or 10 μg of mRNA expressing either the hK2 scFv CAR or an unrelated control CAR. 24 hours after electroporation, CAR surface expression was measured by flow cytometry after staining with PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (upper figure), or PE conjugated with 2 μg / mL biotinylated L protein and streptavidin (lower figure), or PE conjugated with biotinylated hK2 (1 μg / mL) and streptavidin. [Figure 13]This study demonstrates the cytotoxicity of hK2-positive (VCaP, top figure) and hK2-negative (DU145, bottom figure) tumor cells by hK2 CAR-T cells in a 20-hour flow-based assay at a specified effector-to-target cell (E / T) ratio. Target cells were labeled with Cell Trace Violet (CTV) fluorescent dye 24 hours after transient transfection and then co-cultured with hK2 CAR-T cells. Mock T cells served as a negative effector control. Killing percentage was measured as the ratio of the absolute number of viable (vitality dye-negative) target (CTV-positive) cells remaining in the co-culture to the number of viable targets cultured without CAR-T cells. [Figure 14-1] Real-time hK2 CAR-T cell-mediated cytotoxicity is demonstrated. Normalized cell index (CI) plots of VCaP target cells (5E4) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Addition of T cells to target cells mediated cytolysis and subsequent progressive decrease in CI of hK2-positive VCaP cells at E / T ratios from 5:1 to 0.156:1. The decrease in CI values ​​after the addition of effector cells reflected the loss of viability of the target cells. The Y-axis shows normalized CI created by RTCA software and displayed in real time. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 14-2]Real-time hK2 CAR-T cell-mediated cytotoxicity is demonstrated. Normalized cell index (CI) plots of VCaP target cells (5E4) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Addition of T cells to target cells mediated cytolysis and subsequent progressive decrease in CI of hK2-positive VCaP cells at E / T ratios from 5:1 to 0.156:1. The decrease in CI values ​​after the addition of effector cells reflected the loss of viability of the target cells. The Y-axis shows normalized CI created by RTCA software and displayed in real time. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 14-3] Real-time hK2 CAR-T cell-mediated cytotoxicity is demonstrated. Normalized cell index (CI) plots of VCaP target cells (5E4) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Addition of T cells to target cells mediated cytolysis and subsequent progressive decrease in CI of hK2-positive VCaP cells at E / T ratios from 5:1 to 0.156:1. The decrease in CI values ​​after the addition of effector cells reflected the loss of viability of the target cells. The Y-axis shows normalized CI created by RTCA software and displayed in real time. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 15-1]This shows the lack of real-time hK2 CAR-T cell-mediated cytotoxicity in target cells that do not express hK2. Normalized cell index (CI) plots of DU145 target cells (5E3) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Adding T cells to target cells did not decrease CI after addition, and neither hK2 CAR-T nor control CAR-T cells showed cytolytic activity. The Y-axis shows normalized CI generated and displayed in real time by RTCA software. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 15-2] This shows the lack of real-time hK2 CAR-T cell-mediated cytotoxicity in target cells that do not express hK2. Normalized cell index (CI) plots of DU145 target cells (5E3) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Adding T cells to target cells did not decrease CI after addition, and neither hK2 CAR-T nor control CAR-T cells showed cytolytic activity. The Y-axis shows normalized CI generated and displayed in real time by RTCA software. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 15-3]This shows the lack of real-time hK2 CAR-T cell-mediated cytotoxicity in target cells that do not express hK2. Normalized cell index (CI) plots of DU145 target cells (5E3) incubated with mock, 10 μg mRNA electroporated (24 hours after transfection) hK2 11B6 CAR LH, or control CAR-T cells for approximately 72 hours at various E:T ratios. When seeded alone, target cells adhered to the plate, proliferated, and increased CI readout. Adding T cells to target cells did not decrease CI after addition, and neither hK2 CAR-T nor control CAR-T cells showed cytolytic activity. The Y-axis shows normalized CI generated and displayed in real time by RTCA software. The X-axis represents cell culture time and processing time (hours). The mean CI is plotted ± standard deviation. [Figure 16] This shows interferon-gamma (IFN-γ) production by antigen-stimulated hK2 CAR-T cells. Supernatants were collected from xCELligence-based killing assays after co-culture for approximately 70 hours (VCAP#5E4, DU145#5E3). hK2 CAR-LH and control CAR-modified T cells secreted IFN-γ during co-culture with hK2-expressing VCAP cells, but not during co-culture with hK2-negative DU145 cells. Mean IFN-γ concentration ± standard deviation (pg / mL) from duplicate cultures is shown. [Figure 17-1] As shown in the figure, the expression of various hK2 CAR constructs (constructs 1-10) in Jurkat cells containing a luciferase gene driven by a transduction-responsive NFAT promoter (JNL cells) transduced into various hK2 CAR constructs is shown. Expression was determined using biotinylated hK2 followed by streptavidin-conjugated PE. [Figure 17-2]As shown in the figure, the expression of various hK2 CAR constructs (constructs 1-10) in Jurkat cells containing a luciferase gene driven by a transduction-responsive NFAT promoter (JNL cells) transduced into various hK2 CAR constructs is shown. Expression was determined using biotinylated hK2 followed by streptavidin-conjugated PE. [Figure 17-3] As shown in the figure, the expression of various hK2 CAR constructs (constructs 1-10) in Jurkat cells containing a luciferase gene driven by a transduction-responsive NFAT promoter (JNL cells) transduced into various hK2 CAR constructs is shown. Expression was determined using biotinylated hK2 followed by streptavidin-conjugated PE. [Figure 18] This shows the binding between CAR1-10 constructs and their allogeneic cell antigens (hK2 on target cells), as detected by luciferase expression in JNL cells upon binding. Transduced JNL cells and untransduced JNL cells (UTD) containing the specified CAR constructs (CAR1-10) were co-cultured with target cell lines (VCaP or DU145 cells), and luciferase activity was measured as luminescence intensity. If the luminescence intensity was 1.5 times higher than that of UTD cells in the presence of antigen-expressing cells, the construct was considered active. [Figure 19-1] The study showed expression of hK2 CAR1-10 or parental 11B6_HL and 11B6_LH on the surface of T cells. Primary human T cells were transduced with 11B6 heat-stabilized and parental scFv CAR lentivirus (multiple infection dose (MOI): 3), and CAR expression was determined 14 days after transduction using biotinylated hK2 (1 μg / mL), followed by streptavidin-conjugated PE. UTD: Non-transduced control. [Figure 19-2]The study showed expression of hK2 CAR1-10 or parental 11B6_HL and 11B6_LH on the surface of T cells. Primary human T cells were transduced with 11B6 heat-stabilized and parental scFv CAR lentivirus (multiple infection dose (MOI): 3), and CAR expression was determined 14 days after transduction using biotinylated hK2 (1 μg / mL), followed by streptavidin-conjugated PE. UTD: Non-transduced control. [Figure 19-3] The study showed expression of hK2 CAR1-10 or parental 11B6_HL and 11B6_LH on the surface of T cells. Primary human T cells were transduced with 11B6 heat-stabilized and parental scFv CAR lentivirus (multiple infection dose (MOI): 3), and CAR expression was determined 14 days after transduction using biotinylated hK2 (1 μg / mL), followed by streptavidin-conjugated PE. UTD: Non-transduced control. [Figure 20] This shows the percentage of tumor cell growth inhibition in hK2-positive VCaP cells at effector:target ratios of 1:1 or 0.5:1 by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 21] This shows the percentage of tumor cell growth inhibition in PC3 cells at an effector:target ratio of 1:1 by T cells transduced with CAR1-10 or parental 11B6_HL or 11B6_LH in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 22-1]This shows cytokine release by hK2 CAR-T cells. The supernatant collected from co-cultures of hK2 CAR-T cells with VCaP cells overnight (approximately 20 hours) at a 1:1 E / T ratio was analyzed using the 13-plex Milliplex Human High Sensitivity T Cell Kit (HSTCMAG28SPMX13). hK2 CAR-T cells secreted cytokines during co-culture with hK2-expressing VCaP cells, but secretion was minimal during co-culture with untransduced T cells (UTDs). The mean cytokine concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. [Figure 22-2] This shows cytokine release by hK2 CAR-T cells. The supernatant collected from co-cultures of hK2 CAR-T cells with VCaP cells overnight (approximately 20 hours) at a 1:1 E / T ratio was analyzed using the 13-plex Milliplex Human High Sensitivity T Cell Kit (HSTCMAG28SPMX13). hK2 CAR-T cells secreted cytokines during co-culture with hK2-expressing VCaP cells, but secretion was minimal during co-culture with untransduced T cells (UTDs). The mean cytokine concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. [Figure 22-3] This shows cytokine release by hK2 CAR-T cells. The supernatant collected from co-cultures of hK2 CAR-T cells with VCaP cells overnight (approximately 20 hours) at a 1:1 E / T ratio was analyzed using the 13-plex Milliplex Human High Sensitivity T Cell Kit (HSTCMAG28SPMX13). hK2 CAR-T cells secreted cytokines during co-culture with hK2-expressing VCaP cells, but secretion was minimal during co-culture with untransduced T cells (UTDs). The mean cytokine concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. [Figure 23]This shows IFN-γ release by hK2 CAR-T cells. Supernatant was collected from hK2 CAR-T cells co-cultured overnight (approximately 20 hours) with VCaP and DU145 (5E4 cells) cells in a 1:1 E / T ratio. hK2 CAR-modified T cells secreted IFN-γ during co-culture with hK2-expressing VCaP cells, but not during co-culture with hK2-negative DU145 cells. The mean IFN-γ concentration ± standard deviation (pg / mL) from the duplicated cultures is shown. CD3 / 28 bead-stimulated T cells and T cells alone were used as positive and negative controls, respectively. [Figure 24A-1] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-2] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-3] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-4] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-5]The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24A-6] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-1] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-2] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-3] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-4] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-5]The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 24B-6] The histograms of flow cytometry data for untransduced T cells labeled with CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25A-1] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25A-2] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25A-3] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-1] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-2]The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-3] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 25B-4] The histograms of flow cytometry data for untransduced T cells labeled with CD25-positive CellTrace Violet (CTV) (only T cells shown in the figure) or CAR-T cells transduced from CAR1-10, after co-culture with VCaP or DU145 cells for 5 days, or after stimulation with CD3 / 28 beads. [Figure 26] This study shows that hK2 CAR-T cells proliferated more robustly than CD3 / 28 bead-positive controls after co-culture with VCaP cells for 5 days. T cells genetically engineered with various CAR constructs exhibited different proliferative activity and different CAR+ T cell counts. CAR+ T cell counts were based on the mean absolute cell count + / - SEM from three technical replicas. [Figure 27-1] This shows the surface expression of CAR17 (KL2B413_HL), CAR18 (KL2B413_LH), CAR19 (KL2B359_HL), and CAR20 (HK2B359_LH) on the surface of primary T cells. The numbers in each histogram represent the percentage of cells expressing the specified CAR. [Figure 27-2] This shows the surface expression of CAR17 (KL2B413_HL), CAR18 (KL2B413_LH), CAR19 (KL2B359_HL), and CAR20 (HK2B359_LH) on the surface of primary T cells. The numbers in each histogram represent the percentage of cells expressing the specified CAR. [Figure 28-1]This shows the surface expression of CAR17(KL2B413_HL), CAR18(KL2B413_LH), CAR19(KL2B359_HL), and CAR20(KL2B359_LH) on the surface of JNL cells. [Figure 28-2] This shows the surface expression of CAR17(KL2B413_HL), CAR18(KL2B413_LH), CAR19(KL2B359_HL), and CAR20(KL2B359_LH) on the surface of JNL cells. [Figure 29] The figure shows the relative light units (RLU) resulting from luciferase expression in JNL cells mediated by the binding of test CAR-T to its allogeneic receptor in various target cells as specified in the figure. JNL cells containing the specified CAR clones and untransduced NJL cells (UTD) were co-cultured with target cell lines (VCaP, LNCaP / hK2, LNCaP, C4-2B, 22Rv1, or DU145 cells), and luciferase activity was measured as luminescence intensity (RLU, relative light units). KL2B413_HL:CAR17, KL2B413_LH:CAR18, HL2B359_HL:CAR19, KL2B359_LH:CAR20. [Figure 30-1] The figures show the percentage of tumor cell growth inhibition of hK2-positive VCaP cells by transduced CAR-T cells using CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 30-2] The figures show the percentage of tumor cell growth inhibition of hK2-positive VCaP cells by transduced CAR-T cells using CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 31] The figures show the percentage of tumor cell growth inhibition in hK2-negative DU145 cells by transduced CAR-T cells of CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure) in a real-time incucyte killing assay to evaluate antigen-dependent cytotoxicity. Tumor cell growth inhibition (%) = (Initial number of surviving target cells - Current number of surviving target cells) / Initial number of surviving cells × 100 (%). [Figure 32] The figures show IFN-γ production by transduced CAR-T cells or untransduced T cells (UTDs) in co-cultures with the cells specified in the figure: CAR17 (KLK2B413HL in the figure), CAR18 (KLK2B413LH in the figure), CAR19 (KLK2B359HL in the figure), and CAR20 (KLK2B359LH in the figure). [Figure 33A-1] Co-culturing CAR-T cells transduced from CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure), along with VCap cells, resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 33A-2] Co-culturing CAR-T cells transduced from CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure), along with VCap cells, resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 33B-1] Co-culturing CAR-T cells transduced from CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure), along with VCap cells, resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 33B-2] Co-culturing CAR-T cells transduced from CAR17 (B413HL in the figure), CAR18 (B413LH in the figure), CAR19 (B359HL in the figure), and CAR20 (B359LH in the figure), along with VCap cells, resulted in an increase in CD107a+hK2-CAR-T+ cells exhibiting immune cell activation and cytotoxic degranulation. However, co-culturing with hK2-negative DU145 cells did not have any effect. [Figure 34A-1] The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 34A-2] The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 34B-1] The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 34B-2]The image shows flow cytometry histograms of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or transduced CAR-T cells (CAR17 (KLB413HL in the figure), CAR18 (KLB413HL in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAp or DU145 cells for 5 days. UTD: Untransduced. [Figure 35] The percentage of proliferating cells in co-cultures of CellTrace Violet (CTV)-labeled untransduced T cells (T cells only in the figure) or CAR-T cells transduced from CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) is shown after co-culturing with VCAP or DU145 cells for 5 days. UTD: Untransduced. [Figure 36A-1] The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 36A-2] The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 36B-1] The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 36B-2]The histograms of flow cytometry results for transduced CTV+CD25+CAR-T cells CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure) after co-culture with VCAP or DU145 cells for 5 days are shown. [Figure 37] The figures show the percentage of non-transduced T cells (UTDs) expressing CTV, or CAR-T cells transduced to CAR17 (KLB413HL in the figure), CAR18 (KLB413LH in the figure), CAR19 (KLB359HL in the figure), and CAR20 (KLB359LH in the figure), after co-culture with VCaP or DU145 (T cells only) for 5 days or stimulation with CD2 / 28 beads. [Figure 38A] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38B] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38C]Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38D] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38E] Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 38F]Figure 38 shows the conjugated paratopes of the selected anti-hK2 antibody and the selected anti-hK2 / CD3 bispecific antibody. Underlined sequences indicate the CDR region, and highlighted sequences indicate the paratope region. Figure 38A discloses sequence numbers 219 and 220 in order of appearance, respectively. Figure 38B discloses sequence numbers 213 and 224 in order of appearance, respectively. Figure 38C discloses sequence numbers 203 and 215 in order of appearance, respectively. Figure 38D discloses sequence numbers 468 and 469 in order of appearance, respectively. Figure 38E discloses sequence numbers 354 and 221 in order of appearance, respectively. Figure 38F discloses sequence numbers 356 and 222 in order of appearance, respectively. [Figure 39A] This report describes the in vivo efficacy and cytokine profiles of KLK2×CD3 bispecific antibodies in a VCaP xenograft mouse model. Three KLK2×CD3 bispecific antibodies were tested at three dose levels: 5 mg / kg, 1 mg / kg, and 0.2 mg / kg. Tumor growth inhibition was plotted based on tumor volume measurements. [Figure 39B-1] This study describes the in vivo efficacy and cytokine profiles of a KLK2 × CD3 bispecific antibody in a VCaP xenograft mouse model. A 100 μL blood sample was collected from animals via post-orbital hemorrhage 6 hours after the first dose. Plasma was separated from the blood sample by high-speed centrifugation. Luminex assays were performed to quantify IFN- and TNF-α concentrations at various KLK2 bispecific doses. [Figure 39B-2] This study describes the in vivo efficacy and cytokine profiles of a KLK2 × CD3 bispecific antibody in a VCaP xenograft mouse model. A 100 μL blood sample was collected from animals via post-orbital hemorrhage 6 hours after the first dose. Plasma was separated from the blood sample by high-speed centrifugation. Luminex assays were performed to quantify IFN- and TNF-α concentrations at various KLK2 bispecific doses.

[0044] (Detailed description) The methods disclosed are those made in connection with the attached drawings, which form part of this disclosure. It can be more easily understood by referring to the detailed explanation. The disclosed method is: Not limited to the specific methods described and / or shown herein, and furthermore, the use of The terms used are for illustrative purposes only and are not used in the claims. Please understand that this is not intended to limit the methods to those listed.

[0045] All patents, published patent applications, and publications referenced herein are by reference to the law. The entire structure may be incorporated in the same manner as described herein.

[0046] When a list is presented, unless otherwise specified, each individual element of that list and its list It should be understood that all combinations of strikes are distinct embodiments. For example, "A, The list of embodiments presented as "B" or "C" is, It is interpreted as including "A or B", "A or C", "B or C", or "A, B, or C". It should be done.

[0047] When used in this specification and the attached "Claims", the singular forms "a", "an", And "the" can refer to multiple things unless the content is specifically indicated otherwise. Therefore, for example, the reference to "a cell" refers to a set of two or more cells. This includes combinations and similar items.

[0048] Transition phrases: "comprising," "consisting essential" The terms "ly of" and "consisting of" are generally accepted in patent terminology. It is intended to imply the meaning of "to be equipped / include (comp "Rising" is synonymous with "includes," "contains," or "characterizes," and is comprehensive or This is not restrictive and does not exclude other elements or methods not listed. (ii) "consisting of" is not specified in the claims. (iii) "Exclude any element, process or component that is not present, and (iii) "essentially from "The specified materials or processes, as well as the "basic and novel features" of the claimed invention" The scope of the patent claims is limited to "those that do not substantially affect (multiple)" the preparations. Embodiments described with respect to "comprising" (or equivalent) are also implemented. In terms of form, those that are described independently in relation to "consisting of" and "essentially consisting of". provide.

[0049] "Approximately" means that a particular value is within the acceptable margin of error as determined by those skilled in the art. This means that the method by which the value is measured or determined, i.e., the limitations of the measurement system, It depends to some extent on the section. Examples or in the context of a particular assay, result, or embodiment. Unless otherwise expressly stated elsewhere in the specification, “about” means “about to” the practice of the art. This means that the result is within the larger of either one standard deviation or a range of up to 5%. ru.

[0050] "Activation," "stimulation," "activated," or "stimulated" refers to the activation marker. This can lead to the expression of - cytokines, cytokine production, or mediation of target cell proliferation or cytotoxicity. This refers to the induction of a change in the biological state of a cell. Cells are activated by a primary stimulus signal. This can happen. Co-stimulatory signals amplify the magnitude of the primary signal and suppress cell death after initial stimulation. This allows for a more durable activation state, and consequently, higher cytotoxicity. This results in "co-stimulatory signals" such as primary signals like TCR / CD3 ligation. In combination with this, the proliferation of T cells and / or NK cells and / or the enhancement of key molecules This refers to a signal that causes preregulation or downregulation.

[0051] "Alternative scaffolds" associate with variable domains that have a high tolerance for higher-order structures. This refers to a single-chain protein framework containing a structured core. The variable domain is a scalar. To allow for diversity to be introduced without compromising the integrity of the fold, the variable domain is They can be genetically engineered and selected to bind to specific antigens.

[0052] "Antibody-dependent cytotoxicity", "Antibody-dependent cell-mediated cytotoxicity", or "ADCC" The Fc gamma receptor (FcγR) expressed in effector cells is used for coating with antibodies. Target cells that are targeted include natural killer cells (NK), monocytes, macrophages, and neutrophils. A mechanism that induces cell death dependent on interaction with effector cells that have lytic activity, such as spheres. It refers to the introduction.

[0053] "Antibody-dependent cell phagocytosis" or "ADCP" refers to the action of macrophages or dendritic cells, etc. This refers to the mechanism by which antibody-coated target cells are eliminated by phagocytic cells. vinegar.

[0054] An "antigen" is an antigen-binding domain or T cell receptor that can mediate an immune response. Any molecule that can be combined (e.g., proteins, peptides, polysaccharides, glycoproteins) This refers to glycolipids, nucleic acids, some of these, or combinations thereof. Exemplary immune responses and Examples include antibody production and activation of immune cells such as T cells, B cells, or NK cells. Antigens are found in tissue samples, tumor samples, cells, or other biological components, organisms, and tans. Subunits of proteins / antigens, whole cells that have been killed or inactivated, or lysates of living organisms. Genes that can be expressed from a sample, can be synthesized from the sample, or the sample It can be refined from a sample.

[0055] An "antigen-binding fragment" or "antigen-binding domain" is a part of a protein that binds to an antigen. It refers to a synthetic polypeptide, an enzymatically available polypeptide, or a genetically modified polypeptide. These may be modified polypeptides, which may include antigen-binding immunoglobulins Some, for example, VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Domain antibodies (dAb) consisting of a fragment of Fv, one VH domain, or one VL domain. ), shark variable IgNAR domain, camelidized VH domain, VHH domain, FR3-C A minimal recognition unit consisting of amino acid residues that mimic the CDR of antibodies, such as the DR3-FR4 region. T, HCDR1, HCDR2, and / or HCDR3, as well as LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind to the antigen, and antigen binding. Examples include multispecific proteins containing fragments. Antigen-binding fragments (such as VH and VL) are The VH and VL domains are linked to each other via a synthetic linker, allowing for expression as separate single strands. When this occurs, the VH / VL domains pair intramolecularly or intermolecularly to form a monovalent antigen-binding domain. For example, forming a single-chain Fv (scFv) or a diabody. It is possible to form various types of single-chain antibody designs. Antigen-binding fragments are also two To genetically engineer heavy-specific and multispecific proteins, single-specific or multispecific Other possible antibodies, proteins, antigen-binding fragments, or alternative scaffolds Even if conjugated.

[0056] The term "antibody" has a broad meaning and can refer to mouse, human, humanized, and chimeric monoclonal antibodies. Monoclonal antibodies containing the body, antigen-binding fragments, bispecific, triplicate, quadruple-specific antibodies, etc. Multispecific antibodies, dimers, tetramers, or multimers, single-chain antibodies, domain antibodies , and any other modification of the immunoglobulin molecule including the antigen-binding site of the required specificity It contains immunoglobulin molecules that have a disulfide bond. Two heavy chains (HC) and two light chains (L) are interconnected. It is composed of C), and their polymers (e.g., IgM). Each heavy chain has a heavy chain variable region. (heavy chain variable region, VH), and heavy chain constant region (domain CH1, Hin It consists of (CH2 and CH3). Each light chain has a light chain variable region (light ch It consists of a variable region (VL) and a constant region (CL) of the light chain. The VH and VL regions have scattered framework regions (FRs) and complementarity determination regions. It can be further classified into a hypervariable region called the CDR region. Each VH and VL is an amino acid powder. From the end toward the carboxyl terminus, the order is as follows: FR1, CDR1, FR2, CDR2, F It consists of three CDR segments and four FR segments arranged as R3, CDR3, and FR4. It is done. Immunoglobulins are classified into five major types depending on the amino acid sequence of the heavy chain constant domain. They can be assigned to classes, namely IgA, IgD, IgE, IgG, and IgM. gA and IgG are isotypes IgA1, IgA2, IgG1, IgG2, and IgG3. It is further subdivided into IgG4. The antibody light chains of any vertebrate species have their constant domain Based on the amino acid sequence of , there are two distinctly different types, namely kappa (κ) and It can be assigned to either B or lambda (λ).

[0057] "Bispecificity" refers to a characteristic of two different antigens or two different epitopes within the same antigen. This refers to molecules (such as antibodies) that bind heterospecifically. Bispecific molecules can bind to other related antigens, for example, To or monkey, for example, crab-eating macaque (Macaca cynomolgus, cyno) or chin Cross-reactivity occurs against the same antigen (homolog) from other species, such as pansies (Pan troglodytes). It may be responsive, or it may bind to epitopes shared between two or more different antigens. ru.

[0058] “Bispecific anti-hK2 / anti-CD3 antibody”, “hk2 / CD3 antibody”, “hk2×CD3 Antibodies, anti-hK2 / anti-CD3 proteins, etc., bind to hk2 and CD3, and , at least one binding domain that specifically binds to hK2 and CD3 This refers to an antibody containing at least one binding domain. It specifically binds to hK2 and CD3. The domain is typically V H / V L They are a pair. The bispecific anti-hk2×CD3 antibody is h It may be monovalent with respect to binding to either k2 or CD3.

[0059] "Cancer" is a widespread disease characterized by the uncontrolled growth of abnormal cells in the body. This refers to a typical group. Uncontrolled cell division and growth result in the form of malignant tumors that invade adjacent tissues. It can cause growth and metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or " "Cancer tissue" may include tumors.

[0060] When used herein, "chimeric antigen receptors (CARs)" are all derived from a single natural antigen. This is a combination not typically found together in proteins, involving an extracellular target-binding domain and a membrane. It is defined as a cell surface receptor that includes a transcatheter domain and an intracellular signaling domain. This involves the extracellular domain and intracellular signaling domain, which are naturally present in a single receptor. It contains receptors that are not found together in proteins. CARs are primarily found in T cells. It is also intended for use with lymphocytes such as natural killer (NK) cells.

[0061] "Complement-dependent cell damage" or "CDC" refers to the Fc effect of a protein bound to a target. The TARD domain binds to complement component C1q and is activated, which then activates the complement cascade. This refers to a mechanism that induces cell death, causing the death of target cells. Complement activation also affects target cells. This can cause the deposition of complement components on the cell surface, leading to the deposition of complement receptors (e.g., CR3) on leukocytes. The binding facilitates CDC.

[0062] The "complementarity-determining region" (CDR) is the antibody region that binds to the antigen. VH has three C There are DRs (HCDR1, HCDR2, HCDR3), and VL has three CDRs (LCD There are R1, LCDR2, and LCDR3. CDRs contain various depictions such as the following. It can be defined using: Kabat (Wu et al. (1970) J Exp Med 132:211-50;Kabat et al.,Sequences o f Proteins of Immunological Interest,5th Ed.Public Health Service,National Institute Tutes of Health, Bethesda, Md., 1991), Choth ia(Chothia et al. (1987) J Mol Biol 196:90 1-17), IMGT (Lefranc et al. (2003) Dev Comp Immunol 27:55-77), and AbM (Martin and Thorn) (ton J Bmol Biol 263:800-15, 1996). Various descriptions and possible The correspondence with the numbering of the variable region is described (for example, Lefranc et al. (20 03)Dev Comp Immunol 27:55-77;Honegger an d Pluckthun, J Mol Biol (2001) 309:657-70;I International ImmunoGeneTics (IMGT) database; See web resources, http: / / www_imgt_org). UCL B Using available programs such as abYsis with usiness PLC, C DR can be depicted. When used herein, the terms "CDR" and "HCDR1" are used. "HCDR2", "HCDR3", "LCDR1", "LCDR2", and "LCD "R3" refers to Kabat, Chothia as described above, unless otherwise explicitly stated in the specification. This includes CDRs as defined by either the IMGT or AbM method.

[0063] "CD3" is part of the multi - molecular T cell receptor (TCR) complex expressed on T cells and consists of homodimers or heterodimers formed from the association of two or four receptor chains: CD3 epsilon, CD3 delta, CD3 zeta, and CD3 gamma, and refers to the antigen. Human CD3 epsilon contains the amino acid sequence of SEQ ID NO: 442. All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human versions of each protein, polypeptide, or protein fragment, unless expressly specified as being from non - human species derived. Thus, "CD3" means human CD3 unless otherwise specified as being from non - human species, e.g., "mouse CD3", "monkey CD3", etc. Throughout this specification, "CD3 - specific" or "specifically binds to CD3" or "anti - CD3 antibody" refers to an antibody that specifically binds to the CD3 - epsilon polypeptide (SEQ ID NO: 442), including an antibody that specifically binds to the CD3 - epsilon extracellular domain (ECD) (SEQ ID NO: 443). CD3 - ε, together with CD3 - γ, - δ, and - ζ, as well as the T cell receptor α / β and γ / δ heterodimers, forms the T cell receptor - CD3 complex . This complex plays an important role in antigen binding that recognizes several intracellular signaling pathways. The CD3 complex mediates signal transduction, resulting in the activation and proliferation of T cells

[0064] CD3 is required for the immune response. <00008,97>

[0065] SEQ ID NO: 442 (human CD3 epsilon) ​​​​​​MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVS ISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDE DHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVC ENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKA KPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY SGLNQRRI

[0066] SEQ ID NO: 443 (human CD3 epsilon extracellular domain) DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPR GSKPEDANFYLYLRARVCENCMEMD

[0067] "Decrease", "reduce", "attenuate", "lower", or "diminish" generally refers to the ability of a test molecule to mediate a lesser response (i.e., downstream effect) when compared to a response mediated by a control or vehicle. Exemplary responses are T cell expansion, T cell activation, or T cell-mediated tumor cell killing, or binding of a protein to its antigen or receptor, enhanced binding to Fcγ, or enhanced Fc effector functions such as enhanced ADCC, CDC, and / or ADCP. A decrease is a statistically significant difference in the measured response between the test molecule and a control (or vehicle), or about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, e.g., 500, 600, 700, 800, 900, or 1000-fold or more (any number greater than 1), ​​​The decrease of integers and decimal points between them (including, for example, 1.5, 1.6, 1.7, 1.8, etc.) This may be a decrease in the measured response, etc.

[0068] "Differentiation" is the process of reducing the ability or proliferation of a cell, or of making a cell more developmentally limited. This refers to a method of transitioning to a specific state.

[0069] "To code" or "to be coding" refers to a nucleotide of a specified sequence (for example, r A biological organism having any of the amino acids in a specified sequence (RNA, tRNA, and mRNA) It functions as a template for synthesizing other polymers and macromolecules in the process. To do, nucleotides in polynucleotides such as genes, cDNA, or mRNA It refers to the inherent properties of a particular sequence, as well as the biological properties that arise therefrom. Therefore, cells Alternatively, in other biological systems, the transcription and translation of mRNA corresponding to genes produce proteins. In that case, the gene, cDNA, or RNA codes for a protein. The coding strand whose sequence is identical to the mRNA sequence, and the transcription template of the gene or cDNA The non-coding strand used as the nucleotide is either the protein or other part of the gene or cDNA. It can be mentioned as encoding the product of.

[0070] "To strengthen," "to promote," "to increase," "to expand," or "to improve" Generally, a greater response is observed when compared to a response mediated by a control or vehicle. This refers to the ability of the test molecule to mediate (i.e., have a downstream effect). An example response is T cell expansion. T cell activation, or T cell-mediated tumor cell killing, or the antigen or reception of a protein. Binding to a body, enhanced binding to Fcγ, or enhanced ADCC, CDC, and / or enhanced Fc effector functions such as ADCP. Enhancement is a statistically significant difference in the measured responses between the test molecule and a control (or vehicle), or an increase of about 1.1, 1.2, 1. 5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30-fold or more, such as 500, 600, 700, 800, 900, or 1000-fold or more (including all integers greater

[0071] than 1 and the decimal points between them, e.g., 1.5, 1.6, 1.7, 1.8, etc.), measured as an increase in the response. An "epitope" refers to the part of an antigen to which an antibody specifically binds. Epitopes typically consist of a surface population of chemically active (e.g., polar, nonpolar, or hydrophobic) moieties, such as amino acids or polysaccharide side chains, and may have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes

[0072] can be composed of continuous and / or discontinuous amino acids that form

[0073] higher-order structural spatial units. In discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen become very close in three-dimensional space due to the folding of the protein molecule. Antibody

[0074] An "expression vector" is a vector containing polynucleotide sequences that can be used to express emotions. Useful in biological systems or reconstituted biological systems to guide the translation of polypeptides being translated This refers to a vector that can be used.

[0075] "dAb" or "dAb fragment" refers to an antibody fragment composed of a VH domain (War d et al., Nature 341:544 546 (1989)).

[0076] "Fab" or "Fab fragment" consists of VH, CH1, VL, and CL domains. This refers to an antibody fragment.

[0077] "F(ab')2" or "F(ab')2 fragment" is a disulfide bridge within the hinge region. This refers to an antibody fragment containing two Fab fragments connected by [a specific mechanism / tool].

[0078] "Fd" or "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains.

[0079] "Fv" or "Fv fragment" refers to the VH domain and VL domain from a single arm of the antibody. This refers to an antibody fragment composed of n. The Fv fragment is the constant region of the Fab(CH1 and CL) region. It lacks. VH and VL in the Fv fragment are joined together by non-covalent interactions. It is retained.

[0080] The "Fc" polypeptide of the dimer Fc consists of two polypeptides that form the dimer Fc domain. It refers to one of the peptides. For example, the FC polypeptide of dimer IgG FC is IgG (Contains CH2 and IgG CH3 constant domain sequences)

[0081] A "full-length antibody" consists of two heavy chains interconnected by disulfide bonds. ,HC) and two light chains (LC), as well as polymers thereof (e.g., Ig It consists of M). Each heavy chain is composed of a variable heavy chain domain (VH) and a constant heavy chain domain. The heavy chain constant domain is composed of subdomains CH1, hinge, CH2, and CH3. Each light chain consists of a variable light chain domain (VL) and a constant light chain domain (CL). H and VL are complementary resolutions scattered throughout the framework region (FR). Further into the highly variable region known as the complementarity determining region (CDR) It can be classified as follows. Each VH and VL is in the following order from the amino terminus to the carboxyl terminus. Introduction: Arranged in FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 It consists of three CDR segments and four FR segments.

[0082] "Genetic modification" involves introducing "external" (i.e., exogenous or extracellular) genes into a host cell. A gene, DNA, or RNA sequence is introduced, and as a result, the host cell receives the introduced gene or sequence. The desired substance is expressed, typically encoded by the introduced gene or sequence. This refers to the production of a protein or enzyme. The introduced gene or sequence is also called "chromo." These are sometimes called "processed" or "foreign" genes or sequences, and include start, terminate, and progenitor genes. Textures such as motors, signals, secretions, or other sequences used by the genetic mechanisms of cells A regulatory sequence or control that is operably linked to a polynucleotide encoding the antigen receptor. The sequence may contain the gene or sequence. The gene or sequence may contain non-functional sequences that do not have a known function. It is fine. Host cells that receive and express introduced DNA or RNA are "genetically "It is being manipulated." DNA or RNA introduced into a host cell is of the same genus as the host cell or These cells may be of any origin, including those of a species, or may originate from a different genus or species.

[0083] "Different species" refers to two or more polymorphs that are not found in the same relationship to each other in the natural world. Refers to a nucleotide or two or more polypeptides.

[0084] "Heterogeneous polynucleotides" encode two or more neoantigens as described herein. This refers to polynucleotides that do not exist in nature.

[0085] "Heterogeneous polypeptide" is a antigen containing two or more neoantigen polypeptides as described herein. This refers to polypeptides that do not exist naturally.

[0086] A "host cell" refers to any cell containing heterologous nucleic acids. An example heterologous nucleic acid is a vector. —(For example, an expression vector).

[0087] A "human antibody" is an antibody that is optimized to produce the minimum possible immune response when administered to a human subject. This refers to modified antibodies. The variable region of human antibodies is derived from the human immunoglobulin sequence. If the antibody contains a constant region or a portion of a constant region, that constant region also contains a human immunoglobulin sequence. It originates from. Human antibodies are derived from human germline immunoglobulins or rearranged in the variable region of human antibodies. When obtained from a system using immunoglobulin genes, the sequence is of human origin. This includes heavy chain variable regions and light chain variable regions. Such exemplary systems are used to dispose of phages. The played human immunoglobulin gene library and human immunoglobulin loci This includes transgenic non-human animals that possess the antibody, such as mice or rats. "Human antibody" Typically, this refers to differences in the systems used to obtain human antibodies and human immunoglobulin gene loci. i. The intentional introduction or substitution of somatic mutations into the framework or CDR, Or both of these factors result in differences in amino acids when compared to immunoglobulins expressed in humans. It contains. Typically, "human antibodies" are human germline immunoglobulins or rearranged For the amino acid sequence encoded by the immunoglobulin gene, The columns are at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, They are 98% or 99% identical. In some cases, "human antibodies" are, for example, Knappik This is described in et al., (2000) J Mol Biol 296:57-86. Consensus framework sequences obtained from human framework sequence analysis, or for example Shi et al., (2010) J Mol Biol 397:385-96 and The human immunoglobulin presented on phages, as described in International Publication No. 2009 / 085462. It may contain synthetic HCDR3 incorporated into the robulin gene library. Antibodies whose CDRs originate from non-human species are not included in the definition of "human antibodies."

[0088] "Humanized antibodies" are those in which at least one CDR is derived from a non-human species and at least one F The framework refers to antibodies derived from human immunoglobulin sequences. Humanized antibodies are frameworks. Because the work can include substitutions, the framework expresses human immunoglobulins. Alternatively, it may not be an exact copy of the human immunoglobulin germline gene sequence.

[0089] "In combination" means using two or more therapeutic agents together as a mixture, or simultaneously as single agents. This means administering the drugs sequentially to the target in any order as monotherapy agents.

[0090] The "intracellular signaling domain" or "cytoplasmic signaling domain" is a molecular cell This refers to the internal part. This is the functional part of the protein that generates second messengers. To regulate cellular activity through established signaling pathways, information within the cell Acts by transmitting information, or by responding to such messenger. It acts by functioning as an ejector. The intracellular signaling domain is CAR Generates signals that promote the immune effector function of contained cells (e.g., CAR-T cells). do.

[0091] "Isolation" means separating a molecule, such as a recombinant cell, from other components of the system in which it is produced, and essentially separating it. Homogeneity of separated and / or purified molecules (e.g., synthetic polynucleotides or polypeptides) In addition to the population, this refers to proteins that have undergone at least one purification or isolation step. "Separated" refers to molecules that substantially do not contain other cellular materials and / or chemical substances, and more High purity, for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It includes molecules isolated to a purity of 98%, 99%, or 100%.

[0092] "Kallikrein-related peptidase 2" or "hK2" is kallikrein-2, granular kallikrein This refers to a known protein also called Lyrein 2 or HK2. hK2 is a preprotoprotein. It is produced as protein and cleaved during protein degradation to produce active proteases. All hK2 isoforms and variants are included under "hK2". The amino acid sequence of the form is GenBank accession number NP_005542.1 It can be searched using NP_001002231.1 and NP_001243009. The amino acid sequence of the full-length human hK2 in SEQ ID NO: 62 is shown. The sequence is derived from the signal peptide. It contains the d (residues 1-18) and the propeptide region (residues 19-24).

[0093] Sequence ID 62 MWDLVLSIALSVGCTGAVPLIQSRIVGGWECEKHSQPWQ VAVYSHGWAHCGGVLVHPQWV LTAAHCLKKNSQVWLGRHNLFEPDTGQRVPVSHSFPHP LYNMSLLKHQSLRPDEDSSHD LMLLRLSEPAKITDVVKVLGLPTQEPALGTTCYASGWGS IEPEEFLRPRSLQCVSLHLLS NDMCARAYSEKVTEFMLCAGLWTGGKDTCGGDSGGPLVC NGVLQGITSWGPEPCALPEKP AVYTKVVHYRKWIKDTIAANP

[0094] "To regulate" means to have a greater response compared to a response mediated by a control or vehicle. Enhanced or reduced test molecules that mediate a response (i.e., downstream effect) It refers to one of the abilities.

[0095] A "monoclonal antibody" is a substantially homogeneous population of antibody molecules (i.e., a population composed of...) The individual antibodies that make up the body may undergo known modifications such as the removal of the C-terminal lysine from the antibody heavy chain. or amino acid isomerization or amide decomposition, methionine oxidation, or asparagine or glycerin This refers to antibodies obtained from (which are identical except for post-translational modifications such as lutamine adiposition). Noclonal antibodies typically bind to a single antigenic epitope. (Bispecific mono) Clonal antibodies bind to two different antigenic epitopes. Monoclonal antibodies, Monoclonal antibodies may exhibit heterogeneous glycosylation within an antibody population. It is often the case that it is multiplely specific, such as bispecificity, and is monovalent, divalent, or polyvalent. That's fine.

[0096] "Multispecificity" refers to two or more different antigens or two or more different epithelial cells within the same antigen. This refers to molecules such as antibodies that specifically bind to a specific antigen. Multispecific molecules are molecules that bind to other related antigens. For example, humans or monkeys, for example, crab-eating macaques (Macaca fascicularis, cyno) Alternatively, the same antigen (homolog) originating from other species such as chimpanzees (Pan troglodytes) In contrast, epitoids that may exhibit cross-reactivity or are shared between two or more different antigens. It can be combined with P.

[0097] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. NK cells are CD16 + CD56 + and / or CD57 + TCR - Possesses a phenotype NK cells are differentiated lymphocytes that undergo "self-lysis" through the activation of specific cytolytic enzymes. Ability to bind to and kill cells that cannot express HC / HLA antigens, tumor cells, or NK cell activation The ability to kill other disease cells that express the receptor ligand, and to stimulate or inhibit the immune response. It is characterized by its ability to release protein molecules called cytokines.

[0098] The terms "operably linked" and similar phrases are used in reference to nucleic acids or amino acids. In combination, the functional relationships between nucleic acid sequences or amino acid sequences arranged in a mutually functional manner. Refers to a connection. For example, a operably connected promoter, enhancer element, or The pun-reading frame, 5' and 3' UTR, and terminator sequence are nucleic acid components. This results in the precise production of offspring (e.g., RNA), and in some cases, the production of polypeptides. That is, it results in the emergence of an open reading frame. Peptides are designed so that the functional domains of the peptide are positioned at appropriate distances from each other, and each domain has an intended purpose. This refers to peptides that impart a specific function.

[0099] The term "paratope" refers to an antibody molecule that contains residues involved in antigen binding and interaction with the antigen. It refers to an area or region. A paratope is a continuous and / or higher-order structural spatial unit that forms a higher-order structural spatial unit. It can be composed of discontinuous amino acids. The paratope of a given antibody can be obtained through various experimental and computational methods. Using this method, it can be defined and characterized in detail at various levels. The experimental method is water Includes elemental / deuterium exchange mass spectrometry (HX-MS). Paratopes are used in the mapping method. It is defined differently depending on the law.

[0100] "Combination of pharmaceuticals" refers to a combination of two or more active ingredients administered together or separately. It refers to se.

[0101] A "pharmaceutical composition" is a composition obtained by combining an active ingredient and a pharmaceutically acceptable carrier. To point.

[0102] A "medically acceptable carrier" or "excipient" is a non-toxic substance to the target and contains no active ingredient. This refers to components in external pharmaceutical compositions. Exemplary pharmaceutically acceptable carriers include buffers and stabilizers. , or a preservative.

[0103] "Polynucleotide" or "nucleic acid" refers to a sugar-phosphate backbone or other equivalent covalent bond structure. This refers to a synthetic molecule containing nucleotide chains that are covalently bonded by chemical processes. cDNA is, A typical example of a polynucleotide is a DNA or RNA molecule. ru.

[0104] "Preventing," "preventing," "prevention," or "preventive measures" for a disease or disability are considered to be countermeasures against This means preventing injuries to elephants.

[0105] "Proliferation" refers to an increase in cell division, whether symmetrical or asymmetrical.

[0106] A "promoter" refers to the smallest sequence required to initiate transcription. Furthermore, each includes an enhancer or repressor element that enhances or suppresses transcription. obtain.

[0107] "Protein" or "polypeptide" are used interchangeably in this specification and refer to peptide bonds. One or more amino acids, each composed of at least two amino acid residues linked together by a compound. This refers to molecules containing polypeptides. Proteins may be monomers, or the same or It may also be a protein complex of two or more different subunits. Less than 50 A Small polypeptide molecules composed of amino acids can be called "peptides." Proteins are heterologous Fusion proteins, glycoproteins, or phosphorylated, acetylated, myristoylated, palmitic Ilation, glycosylation, oxidation, formylation, amidation, citrullination, polyglutamylation , modified by post-translational modifications such as ADP-ribosylation, pegylation, or biotinylation. It may be any protein. The protein can be recombinantly expressed.

[0108] "Recombinant" refers to a polynylal compound prepared, expressed, produced, or isolated by recombinant means. This refers to creotides, polypeptides, vectors, viruses, and other macromolecules.

[0109] "Regulatory element" is any systolic or systolic that controls several aspects of nucleic acid sequence expression. This refers to Lance-acting genetic elements.

[0110] "Recurrent" means that the disease or signs and symptoms of the disease recur after a period of improvement following prior treatment with medication. It refers to resuming something.

[0111] "Intractable" refers to a disease that does not respond to treatment. Intractable diseases are diagnosed before treatment or at the start of treatment. Diseases that may be resistant to treatment or are intractable may become resistant to treatment during treatment.

[0112] "Single-stranded Fv" or "scFv" is a single-stranded Fv containing at least one antibody that includes a light chain variable region (VL). A fusion protein comprising a body fragment and at least one antibody fragment containing a heavy chain variable region (VH). VL and VH are continuously linked via a polypeptide linker, forming a single-stranded poly This refers to a fusion protein that can be expressed as a lipeptide. Unless otherwise specified, When used herein, scFv has the VL and VH variable regions in any order. This is also possible. For example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv may contain VL-linker-VH, or may contain VH-linker-VL.

[0113] The "(scFv)2" or "tandem scFv" or "bis-scFv" fragment is a fusion protein that contains two light chain variable regions (VL) and two heavy chain variable regions (VH), where the two VL and two VH regions are continuously linked via a polypeptide linker and can be expressed as a single-chain polypeptide. The two VL regions and two VH regions fused by the peptide linker form a bivalent molecule VL -linker A -VH -VH A -linker-VL B -linker-VH B and form two binding sites that can simultaneously bind to two different antigens or epitopes.

[0114] "Specifically binds", "specific binding", "specifically bound", or "binds" refers to a proteinaceous molecule binding to an antigen or an epitope within the antigen with a higher affinity than its affinity for other antigens. Typically, the proteinaceous molecule binds to the antigen or the epitope within the antigen with an equilibrium dissociation constant (K d) of about 1×10 M or less, such as about 5×10 -7 M or less, about 1×10 -8 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M or less and binds to the antigen or the epitope within the antigen, and typically, the said K D d d DThis refers to K for binding to nonspecific antigens (e.g., BSA, casein). D Less than At least 100 times smaller. In the context of prostatic neoantigens as described herein, "specific clusters" "Combination" means that a protein molecule is a variant of the wild-type protein in which the prostate neoantigen is a variant. This refers to binding to the prostate neoantigen without binding to the substance in a detectable manner.

[0115] "Subject" includes any human or non-human animal. "Non-human animal" includes any vertebrate Vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, Examples include cattle, chickens, amphibians, and reptiles. The terms "subject" and "patient" are based on the original text. It can be used interchangeably in detailed documents.

[0116] "T cell" and "T lymphocyte" are interchangeable and used synonymously herein. T cells include thymocytes, naive T lymphocytes, memory T cells, immature T lymphocytes, and mature T cells. Includes T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells are T helpers (T h) Cells, for example, T helper 1 (Th1) or T helper 2 (Th2) cells. T cells are helper T cells (HTLs; CD4 + T cells), CD4 + T cells, cytotoxicity sexual T cells (CTL; ​​CD8 + T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + This may be T cells, or any other subset of T cells. Furthermore, "NKT cells" are also included, which not only express semi-invariant αβT cell receptors. Special T cells that also express various molecular markers associated with normal NK cells, such as NK1.1. This refers to a group of cells. NKT cells include NK1.1 + and NK1.1 - , and CD4 + , CD4 - CD8 + Examples include CD8 cells. The TCR of NKT cells is MHC I It is unique in that it recognizes glycolipid antigens presented by the similar molecule CD Id. T cells have the ability to produce cytokines that promote either inflammation or immune tolerance. It may have either a protective effect or a harmful effect. Also, "gamma-delta T cells (γδT cells) This also includes "cells," which are a small subset of T cells that have different TCRs on their surface. This refers to a special group of proteins, specifically two glycoprotein chains named α- and β-TCR chains. Unlike most T cells in which the CR is composed, the TCR in γδ T cells consists of the γ chain and δ It is composed of chains. γδT cells can play a role in immune surveillance and immune regulation. And the CD8, which is an important and robust source for IL-17 + Cytotoxic T cell response It was found to induce the answer. Furthermore, it suppresses abnormal or excessive immune responses and promotes immune tolerance. This also includes "regulatory T cells" or "Treg," which refer to T cells that play a role in regulating T cells. Typically, this is a transcription factor Foxp3-positive CD4 + It is a T cell and also produces IL-10. CD4 + This may also include T cells, specifically Foxp3-negative regulatory T cells.

[0117] In this specification, "therapeutic effective dose" or "effective dose" as used interchangeably refers to the required dosage. This refers to the amount effective in achieving the desired therapeutic outcome over a given period. The therapeutically effective amount is determined by the individual. Factors such as the patient's condition, age, sex, and weight, as well as treatments that elicit the desired response in the individual, are considered. The effectiveness of a combination of therapeutic drugs or therapeutic agents may vary. Exemplary indicators of the combination include, for example, improvement in the patient's health status, reduction in tumor burden, This includes the cessation or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body. Born.

[0118] "Transduction" refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0119] To "treat," "are treating," or "treat" a disease or disorder such as cancer is as follows: This refers to achieving one or more of the following: reducing the severity and / or duration of the disability, or treating the condition. To inhibit the worsening of symptoms characteristic of the disorder, and to inhibit the disorder in subjects who previously had the disorder. To limit or prevent recurrence, or symptoms in subjects who were previously symptomatic for the disorder. To limit or prevent recurrence.

[0120] "Tumor cells" or "cancer cells" are detected in vivo, ex vivo, or through tissue culture. Furthermore, cancerous, precancerous, or transformed cells with spontaneously occurring or introduced phenotypic changes. Refers to cells. These changes do not necessarily involve the incorporation of new genetic material. Transgenicity involves infection by a transformed virus, integration of new genomic nucleic acids, and removal of exogenous nucleic acids. It can also be caused by contamination, and can occur spontaneously or after exposure to carcinogens. This can also lead to mutations in endogenous genes. Transformation / cancer can be performed in vitro, in vitro. Morphological changes, cell immortalization, control of abnormal proliferation, and lesion shape in vo and ex vivo therapies. Growth, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, and preference for nude mice, etc. This is exemplified by the growth of tumors in suitable animal hosts.

[0121] A "variant," "mutant," or "change" is one or more modifications, for example, one or more Substitution, insertion, or deletion results in a polynucleotide different from the reference polypeptide or reference polynucleotide. This refers to lipeptides or polynucleotides.

[0122] Throughout this specification, the numbering of amino acid residues in the constant region of the antibody is as specified herein. Unless otherwise explicitly stated, Kabat et al., Sequences of Proteins of Immunological Interest,5th E d.Public Health Service,National Institute EU Indicators of Health, Bethesda, MD. (1991) Follow Dex's instructions.

[0123] The mutation in the Ig constant region is named as follows: L351Y_F405A_Y4 07V is L351Y, F405A, and Y40 in one immunoglobulin constant region. This refers to the 7V mutation. L351Y_F405A_Y407V / T394W is a single multimer. L351Y, F405A, and Y4 are present in the protein in the first Ig steady-state region. This refers to the 07V mutation and the T394W mutation in the second Ig constant region.

[0124] "VHH" is a single-domain antibody or na This refers to the nobody. VHH single-domain antibodies use the CH1 of the light and heavy chains of the conventional Fab region. The domain is missing.

[0125] composition of a substance hK2-binding antigen-binding domain This disclosure provides an antigen-binding domain that binds to hK2, and an antigen-binding domain that binds to hK2. Includes monospecific and multispecific proteins (especially bispecific proteins), bound to hK2. A chimeric antigen receptor (CAR) containing an antigen-binding domain, which codes for the aforementioned. This paper proposes polynucleotides, vectors, host cells, and methods for preparing and using the aforementioned. The antigen-binding domain that binds to hK2 as identified herein is improved thermal stability. It showed improved properties in terms of sex. The multispecific proteins disclosed herein are T T cell cytotoxicity mediates the activation and proliferation of T cells, It may be particularly effective in increasing phosphate release and / or demonstrating an increased antitumor effect.

[0126] This disclosure relates to an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2). The isolated protein contains an antigen-binding domain that binds to hK2, which is sequence number 11. A single epitope that binds to an epitope on hK2 as described in 1, 112, 113, 114, or 115. It provides release proteins.

[0127] In certain embodiments, the antigen-binding domain that binds to hK2 is sequence numbers 111 and 11 It binds to the epitope on hK2 described in 2.

[0128] In some embodiments, the antigen-binding domain that binds to hK2 is represented by SEQ ID NO: 113, 1 It binds to the epitope on hK2 described in 14 and 115.

[0129] The present invention also relates to an isolated protein comprising an antigen-binding domain that binds to hK2, and anti The body or its antigen-binding fragment contains the hK2 residue KVTEF (SEQ ID NO: 111) or HYRKW (Sequence ID 112) or SHGWAH (Sequence ID 113) or RNHNLFEPEDTGQ The single link is formed within RVP (sequence number 114) or GWGSIEPEE (sequence number 115). The present invention provides release proteins. In certain embodiments, the present invention also provides antigenic binding to hK2. An isolated protein containing an antigen-binding domain, wherein the antigen-binding domain is KVTEF (sequence) Within the epitope having sequences (number 111) and HYRKW (sequence number 112), hK2 It provides an isolated protein to bind to.

[0130] Using an H / D exchange assay, it is possible to determine the residue in hK2 to which the antibody binds. In the H / D exchange assay, recombinantly expressed soluble hK2 is tested for the presence or absence of an antibody. When incubated in deuterated water in the absence of antibodies for a predetermined time, the antibodies Deuterium incorporation occurs at unprotected, replaceable hydrogen atoms, followed by the protein Protease digestion and LC-MS analysis of peptide fragments will be performed. HD / D exchange up This can be done using a known protocol. An exemplary protocol is shown in Example 3. This is described below. In some embodiments, a quenching buffer (e.g., 8M urea, 1M After quenching the H / D exchange mixture by adding TCEP (pH 3.0), allow it to cool to room temperature. Pass the sample through a weighted, immobilized pepsin / FpXIII column (e.g., 600 μL / min). Next Then, load the digestible fragments into a reverse-phase trap column (e.g., 600 μL / min) and desalt them. For example, (600 μL for 1 minute), separate (for example, with a C18 column), and perform mass spectrometry. The analysis is performed using (for example, a capillary temperature of 275°C, a resolution of 150,000, and a mass range) LTQ(TM) Orbitrap Fusion Lu at (m / z)300-1,800 (Using a MOS mass spectrometer (Thermo Fisher Scientific)).

[0131] The present invention also relates to an isolated protein comprising an antigen-binding domain, wherein the antigen of the reference antibody is... The binding domains are from the group consisting of sequence numbers 111, 112, 113, 114, and 115. An antigen-binding fragment that binds to an epitope on hK2 having a selected sequence and binds to hK2. However, the isolated protein competes with the reference antibody disclosed herein for binding to hK2. Provided. In some embodiments, the reference antibody includes: a. HCDR1, HCDR2, and HCDR3 of VH of sequence number 137, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 138; or b. HCDR1, HCDR2, and HCDR3 of VH of sequence number 162, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 163; or c. HCDR1, HCDR2, and HCDR3 of VH of sequence number 164, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 165; or d. HCDR1, HCDR2, and HCDR3 of VH of sequence number 166, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 167; or e. HCDR1, HCDR2, and HCDR3 of VH of sequence number 168, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 169; or f. HCDR1, HCDR2, and HCDR3 of VH of sequence number 204, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 205; or g. HCDR1, HCDR2, and HCDR3 of VH of sequence number 166, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 444.

[0132] In certain such embodiments, the reference antibody is the heavy chain variable region (VH) of SEQ ID NO: 162 Heavy chain complementarity determination regions (HCDR) 1, HCDR2, and HCDR3, and Sequence ID No. 1 Light chain complementarity determination regions (LCDRs) 1, LCDR2, and L of 63 light chain variable regions (VLs) Includes CDR3.

[0133] Test for binding to soluble hK2 sequence numbers 111, 112, 113, 114, or 115. Competition with the reference antibody of the present invention for antibody binding is performed in vitro using a well-known method. It is possible to label hK2 in the presence of an unlabeled reference antibody. The binding of the antibody to the membrane-proximal region of hK2 may be evaluated by ELISA, or by Bi Competition may be demonstrated using the Acore analysis method or flow cytometry. Test antibody The body binds the reference antibody to soluble hK2 by 85% or more, for example, 90% or more, or 95% or less. When inhibiting, the test antibody competes with the reference antibody for binding to hK2.

[0134] This disclosure relates to an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2). The isolated protein contains an antigen-binding domain that binds to hK2, which includes the following: Provides isolated proteins: Heavy chain complementarity determination region (HCDR) 1 of the heavy chain variable region (VH) of sequence number 137, HCD Determination of light chain complementarity of R2, HCDR3, and the light chain variable region (VL) of Sequence ID No. 138 Regions (LCDR)1, LCDR2, and LCDR3; or HCD of VH of sequence number 137 R1, HCDR2, and HCDR3, and LCDR1 of VL of sequence number 138, LCD R2 and LCDR3; or HCDR1, HCDR2, and HC of VH of Sequence ID No. 162 DR3, and LCDR1, LCDR2, and LCDR3 of VL of sequence number 163; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and sequence number 16 LCDR1, LCDR2, and LCDR3 of VL 5; or HC of VH of SEQ ID NO: 166 DR1, HCDR2, and HCDR3, as well as LCDR1 and LCDR1 of the VL of sequence number 167. DR2 and LCDR3; or HCDR1, HCDR2, and H of VH in sequence number 168 CDR3, and LCDR1, LCDR2, and LCDR3 of VL of sequence number 169; also This refers to HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and sequence number 2 LCDR1, LCDR2, and LCDR3 of VL 05. In a particular embodiment, isolated tan The protein contains an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2). Furthermore, the antigen-binding domain that binds to the hK2 is HCDR1 of VH of SEQ ID NO: 162, H CDR2 and HCDR3, and LCDR1, LCDR2, and VL of Sequence ID No. 163. Includes LCDR3.

[0135] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the h The antigen-binding domains that bind to K2 are as follows: HCDR1, HCDR1, HCDR3, LC Provides isolated proteins including DR1, LCDR2, and LCDR3: Sequence numbers 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively. Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0136] In certain embodiments, the disclosure describes an isolated protein containing an antigen-binding domain that binds to hK2. The substance is such that the antigen-binding domain that binds to the hK2 is such that the respective sequence numbers are SEQ ID NOs: 170 and 1 71, 172, 173, 174, and 175 HCDR1, HCDR1, HCDR3, L This provides isolated proteins including CDR1, LCDR2, and LCDR3.

[0137] In another embodiment, the disclosure relates to an isolated protein containing an antigen-binding domain that binds to hK2. The quality is such that the antigen-binding domain that binds to the hK2 is sequence number 194, 19 HCDR1, HCDR1, HCD with sequence numbers 5, 172, 173, 174, and 175 This provides isolated proteins including R3, LCDR1, LCDR2, and LCDR3.

[0138] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the h The antigen-binding domain that binds to K2 is found in SEQ ID NOs: 137, 162, 164, 166, 168 , or VH of 204 and VL of sequence numbers 138, 163, 165, 167, or 169 It provides isolated proteins, including those mentioned above.

[0139] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the h The K2-binding antigen-binding domain provides an isolated protein containing the following: VH of sequence number 137 and VL of sequence number 138; VH of sequence number 162 and VL of sequence number 163; VH of sequence number 164 and VL of sequence number 165; VH of sequence number 166 and VL of sequence number 167; VH of sequence number 168 and VL of sequence number 169; or VH of sequence number 204 and VL of sequence number 205.

[0140] In certain embodiments, the disclosure describes an isolated protein containing an antigen-binding domain that binds to hK2. The substance is such that the antigen-binding domain that binds to hK2 is the VH of SEQ ID NO: 162 and the This provides an isolated protein containing VL in column number 163.

[0141] This disclosure also relates to an isolated protein comprising an antigen-binding domain that binds to hK2, The antigen-binding domain that binds to hK2 is at least 80% (e.g., VH of SEQ ID NO: 162) For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 163 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% It provides isolated proteins, including those mentioned above.

[0142] In some embodiments, the antigen-binding domain that binds to hK2 is, for example, SEQ ID NO: 1 62 VH and at least 80% (e.g., at least 85%, at least 90%, less) It contains VH and VL of SEQ ID NO: 163, which are 95% or at least 99% identical.

[0143] In some embodiments, the antigen-binding domain that binds to hK2 is, for example, SEQ ID NO: 1 VH of 62 and VL of sequence number 163 and at least 80% (for example, at least 85%) Includes VLs that are identical by at least 90%, at least 95%, or at least 99%.

[0144] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. VH is at least 95% identical to H, and VL is at least 95% identical to Sequence ID No. 163. This includes VL.

[0145] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. VH is at least 95% identical to H, and VL is at least 99% identical to Sequence ID No. 163. This includes VL.

[0146] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. VH is at least 99% identical to H, and VL is at least 99% identical to Sequence ID No. 163. This includes VL.

[0147] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. VH is at least 99% identical to H, and VL is at least 95% identical to Sequence ID No. 163. This includes VL.

[0148] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the h The antigen-binding domain that binds to K2 is found in SEQ ID NOs: 133, 134, 308, 316, 324 , containing the amino acid sequence 325, 404, 405, 406, 407, 408, or 409 , providing isolated proteins. In certain embodiments, this disclosure provides antigenic binding to hK2. An isolated protein containing a binding domain, wherein the antigen-binding domain that binds to hK2 is This provides an isolated protein containing the amino acid sequence of SEQ ID NO: 404.

[0149] In another embodiment, the disclosure relates to an isolated protein containing an antigen-binding domain that binds to hK2. The quality is such that the antigen-binding domain that binds to the hK2 is the amino acid sequence of SEQ ID NO: 405. This provides isolated proteins, including [specific protein].

[0150] This disclosure also relates to an isolated protein comprising an antigen-binding domain that binds to hK2, The antigen-binding domain that binds to hK2 has at least 8 amino acid sequences from the sequence number 404. 0% (for example, at least 85%, at least 90%, at least 95%, or less) This provides isolated proteins containing amino acid sequences that are 99% identical.

[0151] This disclosure also relates to an isolated protein comprising an antigen-binding domain that binds to hK2, The antigen-binding domain that binds to hK2 has at least 8 amino acid sequences from the sequence number 405. 0% (for example, at least 85%, at least 90%, at least 95%, or less) This provides isolated proteins containing amino acid sequences that are 99% identical.

[0152] This disclosure also relates to antigen-binding domains that bind to kallikrein-related peptidase 2 (hK2). An isolated protein containing , wherein the antigen-binding domain that binds to hK2 is sequence number An isolated tan containing the heavy chain variable region (VH) of 75 and the light chain variable region (VL) of SEQ ID NO: 74 The protein is provided. Sequence IDs 75 and 74 show the results when compared with the parent antibody hu11B6. In total, it represents the VH and VL amino acid sequences of the genus, which include variants that exhibit improved thermal stability. The genetically modified site that conferred improved thermal stability is residue P41, I in VH. 49, M70, and A88 (residue numbering according to hu11B6_VH in SEQ ID NO: 5) and V S80, L82, A88, and Y91 in L (according to hu11B6_VL of sequence number 2) (The residue was numbered.)

[0153] JPEG2026064989000001.jpg21159

[0154] JPEG2026064989000002.jpg20167

[0155] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the h The antigen-binding domain that binds to K2 is sequence number 4, 5, 6, 139, 159, or 161. Includes VH and VL of sequence numbers 1, 2, 3, 140, or 160, provided that hK2 The antigen-binding domain that binds to it contains both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2. It provides isolated proteins.

[0156] This disclosure also relates to an isolated protein comprising an antigen-binding domain that binds to hK2, The provided isolated protein comprises an antigen-binding domain that binds to hK2, which includes the following: VH of sequence number 4 and VL of sequence number 1; VH of sequence number 4 and VL of sequence number 2; VH of sequence number 4 and VL of sequence number 3; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; VH of sequence number 6 and VL of sequence number 1; VH of sequence number 6 and VL of sequence number 2; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160; VH of sequence number 139 and VL of sequence number 1; VH of sequence number 139 and VL of sequence number 2; VH of sequence number 139 and VL of sequence number 3; VH of sequence number 139 and VL of sequence number 140; VH of sequence number 139 and VL of sequence number 160; VH of sequence number 159 and VL of sequence number 1; VH of sequence number 159 and VL of sequence number 2; VH of sequence number 159 and VL of sequence number 3; VH of sequence number 159 and VL of sequence number 140; VH of sequence number 159 and VL of sequence number 160; VH of sequence number 161 and VL of sequence number 1; VH of sequence number 161 and VL of sequence number 2; VH of sequence number 161 and VL of sequence number 3; VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or VH of sequence number 161 and VL of sequence number 160.

[0157] This disclosure relates to an isolated protein comprising an antigen-binding domain that binds to hK2, wherein the h The antigen-binding domain that binds to K2 is found in sequence numbers 8, 9, 10, 11, 12, 13, and 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 135, 136, 318, 3 We propose an isolated protein containing the amino acid sequence 19, 320, 321, 322, or 323. To provide.

[0158] In some embodiments, the antigen-binding domain that binds to hK2 is scFv.

[0159] In some embodiments, the antigen-binding domain that binds to hK2 is (scFv)2. ru.

[0160] In some embodiments, the antigen-binding domain that binds to hK2 is Fv.

[0161] In some embodiments, the antigen-binding domain that binds to hK2 is Fab.

[0162] In some embodiments, the antigen-binding domain that binds to hK2 is F(ab')2. ru.

[0163] In some embodiments, the antigen-binding domain that binds to hK2 is Fd.

[0164] In some embodiments, the hK2 antigen-binding domain is dAb.

[0165] In some embodiments, the hK2 antigen-binding domain is VHH.

[0166] In certain embodiments, the antigen-binding domain that binds to hK2 is Fab.

[0167] hK2 binding scFv Both the VH domain and VL domain identified herein that bind to hK2 are V The scFv format supports either H-linker-VL or VL-linker-VH orientation. They can be genetically engineered. Furthermore, both the VH and VL domains identified herein are, for example, For example, it can be used to fabricate the following sc(Fv)2 structure: VH-linker-VL- Linker-VL-Linker-VH, VH-Linker-VL-Linker-VH-Linker- VL. VH-linker-VH-linker-VL-linker-VL. VL-linker-VH -Linker-VH-Linker-VL. VL-Linker-VH-Linker-VL-Linker -VH, or VL-linker-VL-linker-VH-linker-VH.

[0168] The VH and VL domains identified herein should be incorporated into the scFv format. The resulting scFv's binding to hK2 and its thermal stability were evaluated using known methods. It is possible. The combination is ProteOn XPR36, Biacore3000, Alternatively, use a KinExA instrument, ELISA, or a competitive binding assay known to those skilled in the art. This can be evaluated by: Binding is performed using purified scFv or E. coli supernatant or expressed sc Evaluation can be performed using lysed cells containing Fv. The ratio of test scFV to hK2 is as follows: Affinity measurements can be obtained when measured under different conditions (e.g., molar osmotic pressure, pH). They can differ. Therefore, affinity and other binding parameters (e.g., K D , K on , K o ff The measurement of thermal stability is typically performed using standardized conditions and standardized buffers. The temperature is high, such as 50°C, 55°C, or 60°C, and the temperature is set for 5 minutes, 10 minutes, 15 minutes, or 2 minutes. The test scFv is heated for a period of time such as 0 minutes, 25 minutes, or 30 minutes, and the hK of the test scFv is measured. This can be evaluated by measuring the binding to 2. Unheated scFv sample and When compared, scFv, which maintains a bond equivalent to that of hK2, is said to be thermally stable. It can be done.

[0169] In recombinant expression systems, the linker is a peptide linker, which is present in any naturally occurring environment. It may contain amino acids. Examples of amino acids that may be present in the linker are Gly, Ser, and Pr. These are o, Thr, Glu, Lys, Arg, Ile, Leu, His, and The. The Kerrs maintain a precise higher-order configuration relative to each other to preserve desired activities, such as binding to hK2. It is necessary to have an appropriate length to connect VH and VL in a way that forms a structure. be.

[0170] The linker can be approximately 5 to 50 amino acids long. In some embodiments, the linker - has an amino acid length of approximately 10 to 40. In some embodiments, the linker is approximately 1 The amino acid length is 0 to 35. In some embodiments, the linker is approximately 10 to 30. This is the length of the amino acids. In some embodiments, the linker is about 10 to 25 amino acids. It is long. In some embodiments, the linker is about 10 to 20 amino acids long. In some embodiments, the linker is about 15-20 amino acids long. In some embodiments, the linker has a length of 6 amino acids. It has a length of 7 amino acids. In some embodiments, the linker has a length of 8 amino acids. In some embodiments, the linker is 9 amino acid length. The linker is 10 amino acids long. In some embodiments, the linker is 11 The length is one amino acid. In some embodiments, the linker has a length of 12 amino acids. In some embodiments, the linker has a length of 13 amino acids. The linker has a length of 14 amino acids. In some embodiments, the linker has a length of 15 amino acids. The length is one amino acid. In some embodiments, the linker has a length of 16 amino acids. In some embodiments, the linker has a length of 17 amino acids. The linker has a length of 18 amino acids. In some embodiments, the linker has a length of 19 amino acids. The length is 1 amino acid. In some embodiments, the linker is 20 amino acid length. In some embodiments, the linker has a length of 21 amino acids. The linker has a length of 22 amino acids. In some embodiments, the linker has a length of 23 amino acids. The length is one amino acid. In some embodiments, the linker has a length of 24 amino acids. In some embodiments, the linker has a length of 25 amino acids. The linker has a length of 26 amino acids. In some embodiments, the linker is 27 The length is one amino acid. In some embodiments, the linker has a length of 28 amino acids. In some embodiments, the linker has a length of 29 amino acids. The linker is 30 amino acids long. In some embodiments, the linker is 31 The length is one amino acid. In some embodiments, the linker has a length of 32 amino acids. In some embodiments, the linker has a length of 33 amino acids. The linker has a length of 34 amino acids. In some embodiments, the linker has a length of 35 amino acids. The length is one amino acid. In some embodiments, the linker has a length of 36 amino acids. In some embodiments, the linker has a length of 37 amino acids. The linker has a length of 38 amino acids. In some embodiments, the linker has a length of 39 amino acids. The length is 1 amino acid. In some embodiments, the linker is 40 amino acid length. . Linkers containing carbides, glycerides and alas, linkers containing alas and serides, and other linkers. It is a flexible linker.

[0171] Other linker sequences are immunoglobulin heavy chain or light chain isotypes. The ring hinge region may include a portion of CL or CH1. Alternatively, polyethylene glycol. (PEG), polypropylene glycol, polyoxyalkylene, or polyethylene glycol Various non-protein polymers, including copolymers of chol and polypropylene glycol. This may be used as a linker. Examples of linkers that can be used are shown in Table 1. (Additional) The linker is described, for example, in International Publication No. 2019 / 060695.

[0172] In some embodiments, scFv is VH, first ri, from the N-terminus to the C-terminus. Includes L1 and VL (VH-L1-VL).

[0173] In some embodiments, scFv has VL, L1, and Includes VH (VL-L1-VH).

[0174] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 7.

[0175] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 76.

[0176] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 77.

[0177] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 78.

[0178] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 79.

[0179] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 80.

[0180] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 81.

[0181] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 82.

[0182] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 83.

[0183] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 84.

[0184] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 85.

[0185] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 86.

[0186] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 87.

[0187] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 88.

[0188] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 89.

[0189] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 90.

[0190] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 91.

[0191] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 92.

[0192] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 93.

[0193] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 94.

[0194] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 95.

[0195] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 96.

[0196] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 97.

[0197] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 98.

[0198] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 99.

[0199] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 100.

[0200] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 101.

[0201] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 102.

[0202] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 103.

[0203] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 104.

[0204] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 105.

[0205] In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 106.

[0206] In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 107.

[0207] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 108.

[0208] [Table 1]

[0209] In certain embodiments, L1 comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0210] In some embodiments, the scFV includes the following: Heavy chain complementarity determination region (HCDR) 1 of the heavy chain variable region (VH) of sequence number 137, HCD Determination of light chain complementarity of R2, HCDR3, and the light chain variable region (VL) of Sequence ID No. 138 Regions (LCDR)1, LCDR2, and LCDR3; or HCD of VH of sequence number 162 R1, HCDR2, and HCDR3, and LCDR1 of VL of sequence number 163, LCD R2 and LCDR3; or HCDR1, HCDR2, and HC of VH of Sequence ID No. 164 DR3, and LCDR1, LCDR2, and LCDR3 of VL of Sequence ID No. 165; or HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and sequence number 16 LCDR1, LCDR2, and LCDR3 of VL 7; or HC of VH of SEQ ID NO: 168 DR1, HCDR2, and HCDR3, as well as LCDR1 and LCDR1 of the VL of sequence number 169. DR2 and LCDR3; or HCDR1, HCDR2, and H of VH in sequence number 204 CDR3, and LCDR1, LCDR2, and LCDR3 of VL of Sequence ID No. 205; also This includes HCDR1, HCDR2, and HCDR3 of VH in sequence number 139, and sequence number 1 40 VL LCDR1, LCDR2, and LCDR3.

[0211] In some embodiments, scFv is the following HCDR1, HCDR1, HCDR3, Includes LCDR1, LCDR2, and LCDR3 Sequence numbers 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively. Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0212] In some embodiments, scFV is sequence numbers 141, 142, 143, 1 44, 145, and 146 HCDR1, HCDR1, HCDR3, LCDDR1, LCD Includes R2 and LCDR3.

[0213] In some embodiments, scFV is sequence numbers 170, 171, 172, 1 73, 174, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCD Includes R2 and LCDR3.

[0214] In some embodiments, scFV is sequence numbers 176, 177, 178, 1 79, 180, and 181 HCDR1, HCDR1, HCDR3, LCDDR1, LCD Includes R2 and LCDR3.

[0215] In some embodiments, scFV is sequence numbers 170, 183, 184, 1 85, 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, LCD Includes R2 and LCDR3.

[0216] In some embodiments, scFV is sequence numbers 188, 189, 190, 1, respectively. 82, 470, and 209 HCDR1, HCDR1, HCDR3, LCDR1, LCD Includes R2 and LCDR3.

[0217] In some embodiments, scFV is sequence numbers 147, 148, 143, 1 44, 145, and 146 HCDR1, HCDR1, HCDR3, LCDDR1, LCD Includes R2 and LCDR3.

[0218] In some embodiments, scFV is sequence numbers 141, 142, 143, 1 44, 145, and 146 HCDR1, HCDR1, HCDR3, LCDDR1, LCD Includes R2 and LCDR3.

[0219] In some embodiments, scFV is sequence numbers 194, 195, 172, 1 73, 174, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCD Includes R2 and LCDR3.

[0220] In some embodiments, scFV is sequence numbers 196, 197, 178, 1 79, 180, 181 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0221] In some embodiments, scFV is sequence numbers 198, 199, 184, 1 85, 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, LCD Includes R2 and LCDR3.

[0222] In some embodiments, scFV is sequence numbers 200, 201, 190, 1, respectively. HCDR1, HCDR1, HCDR3, LCDR1, LCD 91, 192, and 193 Includes R2 and LCDR3.

[0223] In some embodiments, scFV is sequence numbers 216, 217, 218, 1 82, 470, and 209 HCDR1, HCDR1, HCDR3, LCDR1, LCD Includes R2 and LCDR3.

[0224] In some embodiments, scFV is sequence numbers 63, 65, 66, 67, 6 9, and 71 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and L Includes CDR3.

[0225] In some embodiments, scFV is sequence numbers 63, 65, 66, 68, 7, respectively. 0, and 71 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and L Includes CDR3.

[0226] In some embodiments, scFV is sequence numbers 72, 73, 66, 67, 6, respectively. 9, and 71 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and L Includes CDR3.

[0227] In some embodiments, scFV is VH in SEQ ID NO: 137 and V in SEQ ID NO: 138 Includes L.

[0228] In some embodiments, scFV is VH of SEQ ID NO: 162 and V of SEQ ID NO: 163 Includes L.

[0229] In some embodiments, scFV is VH of SEQ ID NO: 164 and V of SEQ ID NO: 165 Includes L.

[0230] In some embodiments, scFV is VH of SEQ ID NO: 166 and V of SEQ ID NO: 167 Includes L.

[0231] In some embodiments, scFV is VH of SEQ ID NO: 168 and V of SEQ ID NO: 169 Includes L.

[0232] In some embodiments, scFV is VH of SEQ ID NO: 204 and V of SEQ ID NO: 205 Includes L.

[0233] In some embodiments, scFV is VH of SEQ ID NO: 159 and V of SEQ ID NO: 160 Includes L.

[0234] In some embodiments, scFV is VH of SEQ ID NO: 161 and V of SEQ ID NO: 140 Includes L.

[0235] In some embodiments, scFV is VH of SEQ ID NO: 139 and V of SEQ ID NO: 140 Includes L.

[0236] In some embodiments, scFV is VH of SEQ ID NO: 159 and V of SEQ ID NO: 140 Includes L.

[0237] In some embodiments, scFv is at least 80% of VH in sequence number 137 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 138 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0238] In some embodiments, scFv is at least 80% of VH in sequence number 162 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 163 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0239] In some embodiments, scFv is at least 80% of VH of sequence number 164 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 165 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0240] In some embodiments, scFv is at least 80% of VH in sequence number 166 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 167 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0241] In some embodiments, scFv is at least 80% of VH in sequence number 168 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 169 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0242] In some embodiments, scFv is at least 80% of VH of sequence number 204 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 205 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0243] In some embodiments, scFv is at least 80% of VH of sequence number 159 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 160 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0244] In some embodiments, scFv is at least 80% of VH in SEQ ID NO: 161 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 140 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0245] In some embodiments, scFv is at least 80% of VH in sequence number 139 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 140 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0246] In some embodiments, scFv is at least 80% of VH of sequence number 159 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 140 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0247] In some embodiments, scFv is, for example, VH of sequence number 162 and at least 8 0% (for example, at least 85%, at least 90%, at least 95%, or less) It also contains VH and VL of sequence number 163, which are 99% identical.

[0248] In some embodiments, scFv is, for example, VH of SEQ ID NO: 162 and SEQ ID NO: 1 63 VL and at least 80% (e.g., at least 85%, at least 90%, less) It includes VLs that are 95% or at least 99% identical.

[0249] In some embodiments, scFv is at least 95% identical to VH of SEQ ID NO: 162. It includes VH, which is at least 95% identical to VL of Sequence ID No. 163.

[0250] In some embodiments, scFv is at least 99% identical to VH of SEQ ID NO: 162. It includes VH, which is at least 95% identical to VL of Sequence ID No. 163.

[0251] In some embodiments, scFv is at least 99% identical to VH of SEQ ID NO: 162. It includes VH, which is at least 99% identical to VL of sequence number 163.

[0252] In some embodiments, scFv is at least 95% identical to VH of SEQ ID NO: 162. It includes VH, which is at least 99% identical to VL of sequence number 163.

[0253] In some embodiments, scFv is sequence numbers 133, 134, 308, 316, 3 The amino acid sequence 24, 325, 404, 405, 406, 407, 408, or 409 include.

[0254] In some embodiments, scFv is sequence numbers 133, 134, 308, 316, 3 The amino acid sequence of 24, 325, 404, 405, 406, 407, 408, or 409 and At least 80% (for example, at least 85%, at least 90%, at least 95%) Or it contains an amino acid sequence that is at least 99% identical.

[0255] In some embodiments, scFv has the amino acid sequence of SEQ ID NO: 404 and at least 8 0% (for example, at least 85%, at least 90%, at least 95%, or less) It also contains amino acid sequences that are 99% identical.

[0256] In some embodiments, scFv has the amino acid sequence of SEQ ID NO: 405 and at least 8 0% (for example, at least 85%, at least 90%, at least 95%, or less) They also contain amino acid sequences that are 99% identical.

[0257] In some embodiments, scFV has VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74 include.

[0258] In some embodiments, scFV is VH of SEQ ID NOs. 4, 5, or 6 and SEQ ID NOs. 1 , including 2 or 3 VLs.

[0259] In some embodiments, scFV is sequence number 4, 5, 6, 139, 159, or 1 Includes VH 61 and VL of sequence numbers 1, 2, 3, 140, or 160, provided that hK2 The antigen-binding domain that binds to it includes both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2. That's not true.

[0260] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1. .

[0261] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2 .

[0262] In some embodiments, scFV includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3. .

[0263] In some embodiments, scFV has VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140 include.

[0264] In some embodiments, scFV has VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160 include.

[0265] In some embodiments, scFV includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1 .

[0266] In some embodiments, scFV includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3. .

[0267] In some embodiments, scFV has VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140 include.

[0268] In some embodiments, scFV has VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160 include.

[0269] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1. .

[0270] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2 .

[0271] In some embodiments, scFV includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3. .

[0272] In some embodiments, scFV has VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140 include.

[0273] In some embodiments, scFV has VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160 include.

[0274] In some embodiments, scFV has VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1 include.

[0275] In some embodiments, scFV has VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2 include.

[0276] In some embodiments, scFV has VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3 include.

[0277] In some embodiments, scFV is VH of SEQ ID NO: 139 and V of SEQ ID NO: 140 Includes L.

[0278] In some embodiments, scFV is VH of SEQ ID NO: 139 and V of SEQ ID NO: 160 Includes L.

[0279] In some embodiments, scFV has VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1 include.

[0280] In some embodiments, scFV has VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2 include.

[0281] In some embodiments, scFV has VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3 include.

[0282] In some embodiments, scFV is VH of SEQ ID NO: 159 and V of SEQ ID NO: 140 Includes L.

[0283] In some embodiments, scFV is VH of SEQ ID NO: 159 and V of SEQ ID NO: 160 Includes L.

[0284] In some embodiments, scFV has VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1 include.

[0285] In some embodiments, scFV has VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2 include.

[0286] In some embodiments, scFV has VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3 include.

[0287] In some embodiments, scFV is VH of SEQ ID NO: 161 and V of SEQ ID NO: 140 Includes L.

[0288] In some embodiments, scFV is VH of SEQ ID NO: 161 and V of SEQ ID NO: 160 Includes L.

[0289] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 8.

[0290] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 9.

[0291] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 10.

[0292] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 11.

[0293] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 12.

[0294] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 13.

[0295] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 14.

[0296] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 15.

[0297] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 16.

[0298] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 17.

[0299] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 18.

[0300] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 19.

[0301] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 20.

[0302] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 21.

[0303] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 22.

[0304] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 23.

[0305] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 135.

[0306] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 136.

[0307] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 318.

[0308] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 319.

[0309] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 320.

[0310] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 321.

[0311] In some embodiments, scFv includes the amino acid sequence of SEQ ID NO: 322.

[0312] In some embodiments, scFv contains the amino acid sequence of SEQ ID NO: 323.

[0313] hK2-binding and other antigen-binding domains Both the VH domain and VL domain identified herein that bind to hK2 are F It is possible to genetically modify the ab, F(ab')2, Fd, or Fv format, The binding and thermal stability of these molecules to hK2 should be evaluated using the assay described herein. It is possible.

[0314] In some embodiments, Fab includes the following: Heavy chain complementarity determination region (HCDR) 1 of the heavy chain variable region (VH) of sequence number 137, HCD Determination of light chain complementarity of R2, HCDR3, and the light chain variable region (VL) of Sequence ID No. 138 Area (LCDR)1, LCDR2, and LCDR3; or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 162, and SEQ ID NO: 1 63 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 164, and SEQ ID NO: 1 65 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 166, and SEQ ID NO: 1 67 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 168, and SEQ ID NO: 1 69 VL LCDR1, LCDR2, and LCDR3; or Or HCDR1, HCDR2, and HCDR3 of VH of sequence number 204, and sequence number LCDR1, LCDR2, and LCDR3 of unit 205VL.

[0315] In a particular embodiment, Fab is HCDR1, HCDR2, and VH of Sequence ID No. 162. HCDR3, and LCDR1, LCDR2, and LCDR3 of VL of Sequence ID No. 163 Includes.

[0316] In some embodiments, Fab is HCDR1, HCDR1, HCDR3, L Includes CDR1, LCDR2, and LCDR3 Sequence numbers 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0317] In some embodiments, Fab is sequence numbers 141, 142, 143, and 14 4, 145, and 146 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0318] In some embodiments, Fab is sequence numbers 170, 171, 172, and 17 3, 174, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0319] In some embodiments, Fab is sequence numbers 176, 177, 178, and 17 9, 180, and 181 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0320] In some embodiments, Fab is sequence numbers 170, 183, 184, and 18, respectively. 5, 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0321] In some embodiments, Fab is sequence numbers 188, 189, 190, and 19, respectively. HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0322] In some embodiments, Fab is sequence numbers 206, 207, 208, and 18, respectively. 2,470,209 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and includes LCDR3.

[0323] In some embodiments, Fab is sequence numbers 147, 148, 143, and 14 4, 145, and 146 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0324] In some embodiments, Fab is sequence numbers 194, 195, 172, and 17, respectively. 3, 174, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0325] In some embodiments, Fab is sequence numbers 196, 197, 178, and 17, respectively. 9, 180, 181 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and includes LCDR3.

[0326] In some embodiments, Fab is sequence numbers 198, 199, 184, and 18, respectively. 5, 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0327] In some embodiments, Fab is sequence numbers 200, 201, 190, and 19, respectively. HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0328] In some embodiments, Fab is sequence numbers 216, 217, 218, and 18, respectively. 2,470, and 209 HCDR1, HCDR1, HCDR3, LCDR1, LCDR Includes 2, and LCDR3.

[0329] In a particular embodiment, Fab is sequence numbers 170, 171, 172, 173, respectively. 174 and 175, or sequence numbers 194, 195, 172, 173, and 174 respectively, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LC Includes DR3.

[0330] In some embodiments, Fab is VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138 Includes.

[0331] In some embodiments, Fab is VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163 Includes.

[0332] In some embodiments, Fab is VH of SEQ ID NO: 164 and VL of SEQ ID NO: 165 Includes.

[0333] In some embodiments, Fab is VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167 Includes.

[0334] In some embodiments, Fab is VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169 Includes.

[0335] In some embodiments, Fab is VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205 Includes.

[0336] In some embodiments, Fab includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74 nothing.

[0337] In certain embodiments, Fab includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163 nothing.

[0338] In some embodiments, scFv is at least 80% of VH in sequence number 162 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 163 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0339] In some embodiments, scFv is, for example, VH of sequence number 162 and at least 8 0% (for example, at least 85%, at least 90%, at least 95%, or less) It also contains VH and VL of sequence number 163, which are 99% identical.

[0340] In some embodiments, scFv is, for example, VH of SEQ ID NO: 162 and SEQ ID NO: 1 63 VL and at least 80% (e.g., at least 85%, at least 90%, less) It includes VLs that are 95% or at least 99% identical.

[0341] In some embodiments, Fab is at least 95% identical to VH of SEQ ID NO: 162. Includes a VH and a VL that is at least 95% identical to VL of sequence number 163.

[0342] In some embodiments, Fab is at least 99% identical to VH of SEQ ID NO: 162. Includes a VH and a VL that is at least 95% identical to VL of sequence number 163.

[0343] In some embodiments, Fab is at least 99% identical to VH of SEQ ID NO: 162. Includes a VH and a VL that is at least 99% identical to VL of sequence number 163.

[0344] In some embodiments, Fab is at least 99% identical to VH of SEQ ID NO: 162. Includes a VH and a VL that is at least 95% identical to VL of sequence number 163.

[0345] In some embodiments, scFv is at least 80% of VH in sequence number 137 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 138 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0346] In some embodiments, scFv is at least 80% of VH of sequence number 164 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 165 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0347] In some embodiments, scFv is at least 80% of VH in sequence number 166 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 167 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0348] In some embodiments, scFv is at least 80% of VH in sequence number 168 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 169 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0349] In some embodiments, scFv is at least 80% of VH of sequence number 204 (e.g., For example, at least 85%, at least 90%, at least 95%, or at least 99% ) VH and VL of sequence number 205 are identical and at least 80% (for example, at least 8 VLs that are identical by 5%, at least 90%, at least 95%, or at least 99% include.

[0350] In some embodiments, Fab is sequence number 4, 5, 6, 139, 159, or 16 Includes VH of 1 and VL of sequence numbers 1, 2, 3, 140, or 160, provided that Fab is It will not contain both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2.

[0351] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1.

[0352] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2.

[0353] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3.

[0354] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140 nothing.

[0355] In some embodiments, Fab includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160 nothing.

[0356] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1.

[0357] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3.

[0358] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140 nothing.

[0359] In some embodiments, Fab includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160 nothing.

[0360] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1.

[0361] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2.

[0362] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3.

[0363] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140. nothing.

[0364] In some embodiments, Fab includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160 nothing.

[0365] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1. nothing.

[0366] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2 nothing.

[0367] In some embodiments, Fab includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3 nothing.

[0368] In some embodiments, Fab is VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140 Includes.

[0369] In some embodiments, Fab is VH of SEQ ID NO: 139 and VL of SEQ ID NO: 160 Includes.

[0370] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1. nothing.

[0371] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2 nothing.

[0372] In some embodiments, Fab includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3 nothing.

[0373] In some embodiments, Fab is VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140 Includes.

[0374] In some embodiments, Fab is VH of SEQ ID NO: 159 and VL of SEQ ID NO: 160 Includes.

[0375] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1. nothing.

[0376] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2 nothing.

[0377] In some embodiments, Fab includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3 nothing.

[0378] In some embodiments, Fab is VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140 Includes.

[0379] In some embodiments, Fab is VH of SEQ ID NO: 161 and VL of SEQ ID NO: 160 Includes.

[0380] VH and VL of Fab, which contain an antigen-binding domain that binds to hK2, are respectively Fab- Fc HC (VH-CH1-Hinge-CH2-CH3) and Fab-Fc LC (VL- It can be genetically engineered in the CL) format. In certain such embodiments, Fab-F c LC is at least 80% (for example, at least 85%, and at least) of SEQ ID NO: 354. It also contains amino acid sequences that are 90%, at least 95%, or at least 99% identical. In certain embodiments, Fab-Fc HC has the same amino acid sequence as SEQ ID NO: 354. Includes.

[0381] In some embodiments, Fab-Fc HC has a C-terminal lysine residue (e.g., K47 7) includes. In some embodiments, Fab-Fc HC has a C-terminal lysine residue. The amino acid sequence, and at least 80% (for example, at least 85%) of SEQ ID NO: 361. Contains sequences that are identical by at least 90%, at least 95%, or at least 99%. In certain embodiments, Fab-Fc HC contains the amino acid sequence of SEQ ID NO: 361.

[0382] In some embodiments, Fab-Fc LC is at least 80% of SEQ ID NO: 221 (For example, at least 85%, at least 90%, at least 95%, or at least 9 9%) Contains identical amino acid sequences. In certain embodiments, Fab-Fc LC contains Contains an amino acid sequence identical to that of column number 221.

[0383] As shown in the examples, hK2 is bound to incorporate into a multispecific construct. A particularly preferred antigen-binding domain is Fab-Fc having the amino acid sequence of SEQ ID NO: 354. It contains Fab-Fc LC having the amino acid sequences of HC and SEQ ID NO: 221.

[0384] In some embodiments, F(ab')2 includes the following: Heavy chain complementarity determination region (HCDR) 1 of the heavy chain variable region (VH) of sequence number 137, HCD Determination of light chain complementarity of R2, HCDR3, and the light chain variable region (VL) of Sequence ID No. 138 Area (LCDR)1, LCDR2, and LCDR3; or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 162, and SEQ ID NO: 1 63 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 164, and SEQ ID NO: 1 65 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 166, and SEQ ID NO: 1 67 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 168, and SEQ ID NO: 1 69 VL LCDR1, LCDR2, and LCDR3; or Or HCDR1, HCDR2, and HCDR3 of VH of sequence number 204, and sequence number LCDR1, LCDR2, and LCDR3 of unit 205VL.

[0385] In some embodiments, F(ab')2 is the following HCDR1, HCDR1, HCDR 3. Including LCDR1, LCDR2, and LCDR3 Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0386] In some embodiments, F(ab')2 is sequence numbers 141, 142, and 14, respectively. 3, 144, 145, and 146 HCDR1, HCDR1, HCDR3, LCDR1, Includes LCDR2 and LCDR3.

[0387] In some embodiments, F(ab')2 is sequence numbers 170, 171, and 17, respectively. 2, 173, 174 and 175 HCDR1, HCDR1, HCDR3, LCDR1, L Includes CDR2 and LCDR3.

[0388] In some embodiments, F(ab')2 is sequence numbers 176, 177, and 177, respectively. 8, 179, 180 and 181 HCDR1, HCDR1, HCDR3, LCDR1, L Includes CDR2 and LCDR3.

[0389] In some embodiments, F(ab')2 is sequence numbers 170, 183, and 18, respectively. 4, 185, 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, Includes LCDR2 and LCDR3.

[0390] In some embodiments, F(ab')2 is sequence numbers 188, 189, and 19, respectively. HCDR1, HCDR1, HCDR3, LCDR1, 0, 191, 192, and 193 Includes LCDR2 and LCDR3.

[0391] In some embodiments, F(ab')2 is sequence numbers 206, 207, and 20, respectively. 8, 182, 470, 209 HCDR1, HCDR1, HCDR3, LCDR1, LC Includes DR2 and LCDR3.

[0392] In some embodiments, F(ab')2 is sequence numbers 147, 148, and 14, respectively. 3, 144, 145, and 146 HCDR1, HCDR1, HCDR3, LCDR1, Includes LCDR2 and LCDR3.

[0393] In some embodiments, F(ab')2 is sequence numbers 194, 195, and 17, respectively. 2, 173, 174, and 175 HCDR1, HCDR1, HCDR3, LCDR1, Includes LCDR2 and LCDR3.

[0394] In some embodiments, F(ab')2 is sequence numbers 196, 197, and 17, respectively. 8, 179, 180, 181 HCDR1, HCDR1, HCDR3, LCDR1, LC Includes DR2 and LCDR3.

[0395] In some embodiments, F(ab')2 is sequence numbers 198, 199, and 18, respectively. 4, 185, 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, Includes LCDR2 and LCDR3.

[0396] In some embodiments, F(ab')2 is sequence numbers 200, 201, and 19, respectively. HCDR1, HCDR1, HCDR3, LCDR1, 0, 191, 192, and 193 Includes LCDR2 and LCDR3.

[0397] In some embodiments, F(ab')2 is sequence numbers 216, 217, and 21, respectively. 8, 182, 470, and 209 HCDR1, HCDR1, HCDR3, LCDR1, Includes LCDR2 and LCDR3.

[0398] In some embodiments, F(ab')2 is VH of SEQ ID NO: 137 and SEQ ID NO: 13 Includes 8 VLs.

[0399] In some embodiments, F(ab')2 is VH of SEQ ID NO: 162 and SEQ ID NO: 16 Includes 3 VLs.

[0400] In some embodiments, F(ab')2 is VH of SEQ ID NO: 164 and SEQ ID NO: 16 Includes 5 VL.

[0401] In some embodiments, F(ab')2 is VH of SEQ ID NO: 166 and SEQ ID NO: 16 Includes 7 VL.

[0402] In some embodiments, F(ab')2 is VH of SEQ ID NO: 168 and SEQ ID NO: 16 Includes 9 VLs.

[0403] In some embodiments, F(ab')2 is VH of SEQ ID NO: 204 and SEQ ID NO: 20 Includes 5 VL.

[0404] In some embodiments, F(ab')2 is VH of SEQ ID NO: 75 and SEQ ID NO: 74 Includes VL.

[0405] In some embodiments, F(ab')2 is sequence numbers 4, 5, 6, 139, 159, or including VH of 161 and VL of sequence numbers 1, 2, 3, 140, or 160, however, Fab does not contain both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2.

[0406] In some embodiments, F(ab')2 is VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1 Includes.

[0407] In some embodiments, F(ab')2 is VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2 Includes.

[0408] In some embodiments, F(ab')2 is VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3 Includes.

[0409] In some embodiments, F(ab')2 is VH of SEQ ID NO: 4 and SEQ ID NO: 140 Includes VL.

[0410] In some embodiments, F(ab')2 is VH of SEQ ID NO: 4 and SEQ ID NO: 160 Includes VL.

[0411] In some embodiments, F(ab')2 is VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1 Includes.

[0412] In some embodiments, F(ab')2 is VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3 Includes.

[0413] In some embodiments, F(ab')2 is VH of SEQ ID NO: 5 and SEQ ID NO: 140 Includes VL.

[0414] In some embodiments, F(ab')2 is VH of SEQ ID NO: 5 and SEQ ID NO: 160 Includes VL.

[0415] In some embodiments, F(ab')2 is VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1 Includes.

[0416] In some embodiments, F(ab')2 is VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2 Includes.

[0417] In some embodiments, F(ab')2 is VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3 Includes.

[0418] In some embodiments, F(ab')2 is VH of SEQ ID NO: 6 and SEQ ID NO: 140 Includes VL.

[0419] In some embodiments, F(ab')2 is VH of SEQ ID NO: 6 and SEQ ID NO: 160 Includes VL.

[0420] In some embodiments, F(ab')2 is VH of SEQ ID NO: 139 and SEQ ID NO: 1 Includes VL.

[0421] In some embodiments, F(ab')2 is VH of SEQ ID NO: 139 and SEQ ID NO: 2 Includes VL.

[0422] In some embodiments, F(ab')2 is VH of SEQ ID NO: 139 and SEQ ID NO: 3 Includes VL.

[0423] In some embodiments, F(ab')2 is VH of SEQ ID NO: 139 and SEQ ID NO: 14 Includes VL of 0.

[0424] In some embodiments, F(ab')2 is VH of SEQ ID NO. 139 and SEQ ID NO. 16 Includes VL of 0.

[0425] In some embodiments, F(ab')2 is VH of SEQ ID NO: 159 and SEQ ID NO: 1 Includes VL.

[0426] In some embodiments, F(ab')2 is VH of SEQ ID NO: 159 and SEQ ID NO: 2 Includes VL.

[0427] In some embodiments, F(ab')2 is VH of SEQ ID NO: 159 and SEQ ID NO: 3 Includes VL.

[0428] In some embodiments, F(ab')2 is VH of SEQ ID NO: 159 and SEQ ID NO: 14 Includes VL of 0.

[0429] In some embodiments, F(ab')2 is VH of SEQ ID NO: 159 and SEQ ID NO: 16 Includes VL of 0.

[0430] In some embodiments, F(ab')2 is VH of SEQ ID NO: 161 and SEQ ID NO: 1 Includes VL.

[0431] In some embodiments, F(ab')2 is VH of SEQ ID NO: 161 and SEQ ID NO: 2 Includes VL.

[0432] In some embodiments, F(ab')2 is VH of SEQ ID NO: 161 and SEQ ID NO: 3 Includes VL.

[0433] In some embodiments, F(ab')2 is VH of SEQ ID NO: 161 and SEQ ID NO: 14 Includes VL of 0.

[0434] In some embodiments, F(ab')2 is VH of SEQ ID NO: 161 and SEQ ID NO: 16 Includes VL of 0.

[0435] In some embodiments, Fv includes the following: Heavy chain complementarity determination region (HCDR) 1 of the heavy chain variable region (VH) of sequence number 137, HCD Determination of light chain complementarity of R2, HCDR3, and the light chain variable region (VL) of Sequence ID No. 138 Area (LCDR)1, LCDR2, and LCDR3; or HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 162, and SEQ ID NO: 1 63 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 164, and SEQ ID NO: 1 65 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 166, and SEQ ID NO: 1 67 VL LCDR1, LCDR2, and LCDR3; HCDR1, HCDR2, and HCDR3 of VH of SEQ ID NO: 168, and SEQ ID NO: 1 69 VL LCDR1, LCDR2, and LCDR3; or Or HCDR1, HCDR2, and HCDR3 of VH of sequence number 204, and sequence number LCDR1, LCDR2, and LCDR3 of unit 205VL.

[0436] In some embodiments, Fv is the following HCDR1, HCDR1, HCDR3, LC Includes DR1, LCDR2, and LCDR3 Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0437] In some embodiments, Fv is sequence numbers 141, 142, 143, and 144, respectively. , 145, and 146 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0438] In some embodiments, Fv is represented by sequence numbers 170, 171, 172, and 173, respectively. , 174, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0439] In some embodiments, Fv is represented by sequence numbers 176, 177, 178, and 179, respectively. , 180, and 181 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0440] In some embodiments, Fv is sequence numbers 170, 183, 184, and 185, respectively. , 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0441] In some embodiments, Fv is sequence numbers 188, 189, 190, and 191, respectively. , 192, and 193 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0442] In some embodiments, Fv is sequence numbers 206, 207, 208, and 182, respectively. , 470, 209 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and Includes LCDR3.

[0443] In some embodiments, Fv is represented by sequence numbers 147, 148, 143, and 144, respectively. , 145, and 146 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0444] In some embodiments, Fv is sequence numbers 194, 195, 172, and 173, respectively. , 174, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0445] In some embodiments, Fv is represented by sequence numbers 196, 197, 178, and 179, respectively. , 180, 181 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and Includes LCDR3.

[0446] In some embodiments, Fv is sequence numbers 198, 199, 184, and 185, respectively. , 186, and 187 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0447] In some embodiments, Fv is sequence numbers 200, 201, 190, and 191, respectively. , 192, and 193 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0448] In some embodiments, Fv is sequence numbers 216, 217, 218, and 182, respectively. , 470, and 209 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2 , and LCDR3.

[0449] In some embodiments, Fv is VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138 include.

[0450] In some embodiments, Fv is VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163 include.

[0451] In some embodiments, Fv is VH of sequence number 164 and VL of sequence number 165 include.

[0452] In some embodiments, Fv is VH of sequence number 166 and VL of sequence number 167 include.

[0453] In some embodiments, Fv is VH of sequence number 168 and VL of sequence number 169 include.

[0454] In some embodiments, Fv is VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205 include.

[0455] In some embodiments, Fv includes VH of SEQ ID NO: 75 and VL of SEQ ID NO: 74. .

[0456] In some embodiments, Fv is VH of sequence numbers 4, 5, or 6 and sequence numbers 1, 2 , or including 3 VLs.

[0457] In some embodiments, Fv is sequence number 4, 5, 6, 139, 159, or 161 Includes VH and VL of sequence numbers 1, 2, 3, 140, or 160, provided that Fab is It is not possible to include both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2.

[0458] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1.

[0459] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2.

[0460] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3.

[0461] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140. .

[0462] In some embodiments, Fv includes VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160. .

[0463] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1.

[0464] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3.

[0465] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140. .

[0466] In some embodiments, Fv includes VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160. .

[0467] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1.

[0468] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 2.

[0469] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3.

[0470] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140. .

[0471] In some embodiments, Fv includes VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160. .

[0472] In some embodiments, Fv includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 1. .

[0473] In some embodiments, Fv includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 2. .

[0474] In some embodiments, Fv includes VH of SEQ ID NO: 139 and VL of SEQ ID NO: 3. .

[0475] In some embodiments, Fv is VH of SEQ ID NO: 139 and VL of SEQ ID NO: 140 include.

[0476] In some embodiments, Fv is VH of sequence number 139 and VL of sequence number 160. include.

[0477] In some embodiments, Fv includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 1. .

[0478] In some embodiments, Fv includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 2. .

[0479] In some embodiments, Fv includes VH of SEQ ID NO: 159 and VL of SEQ ID NO: 3. .

[0480] In some embodiments, Fv is VH of SEQ ID NO: 159 and VL of SEQ ID NO: 140 include.

[0481] In some embodiments, Fv is VH of sequence number 159 and VL of sequence number 160. include.

[0482] In some embodiments, Fv includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 1. .

[0483] In some embodiments, Fv includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 2. .

[0484] In some embodiments, Fv includes VH of SEQ ID NO: 161 and VL of SEQ ID NO: 3. .

[0485] In some embodiments, Fv is VH of sequence number 161 and VL of sequence number 140. include.

[0486] In some embodiments, Fv is VH of sequence number 161 and VL of sequence number 160 include.

[0487] In some embodiments, Fd includes VH of sequence number 75.

[0488] In some embodiments, Fd includes VH of sequence number 4.

[0489] In some embodiments, Fd includes VH of sequence number 5.

[0490] In some embodiments, Fd includes VH of sequence number 6.

[0491] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 210 Includes HC and LC of SEQ ID NO: 221.

[0492] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 211 Includes HC and LC of SEQ ID NO: 222.

[0493] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 212 Includes HC and LC of SEQ ID NO: 223.

[0494] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 213 Includes HC and LC of sequence number 224.

[0495] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 219 Includes HC and LC of SEQ ID NO: 220.

[0496] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 354 and at least 80% (for example, at least 85%, at least 90%, at least 95%) , or at least 99%) identical to HC and SEQ ID NO: 221 and at least 80% (for example) (For example, at least 85%, at least 90%, at least 95%, or at least 99%) Includes identical LCs.

[0497] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 354 and at least 80% (for example, at least 85%, at least 90%, at least 95%) , or containing HC and LC of SEQ ID NO: 221 which are identical (at least 99%).

[0498] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 354 HC and SEQ ID NO: 221 and at least 80% (e.g., at least 85%, at least Includes LCs that are 90%, at least 95%, or at least 99% identical.

[0499] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 354 HC is at least 95% identical to and L is at least 95% identical to sequence number 221. Includes C.

[0500] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 354 HC is at least 99% identical to sequence number 221 and L is at least 95% identical to sequence number 221. Includes C.

[0501] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 354 HC is at least 99% identical to sequence number 221 and L is at least 99% identical to sequence number 221. Includes C.

[0502] In some embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is SEQ ID NO: 354 HC is at least 95% identical to sequence number 221 and L is at least 99% identical to sequence number 221. Includes C.

[0503] In certain embodiments, the isolated anti-hK2 antibody or its antigen-binding fragment is H of SEQ ID NO: 354 Includes C and LC of sequence number 221.

[0504] Homologous antigen-binding domains and antigen-binding domains with conserved substitutions Variants of the antigen-binding domain that bind to hK2 are within the scope of this disclosure. For example, Does the variant retain improved functional properties compared to the parent antigen-binding domain? Or, insofar as it is present, the antigen-binding domain that binds to hK2 contains 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2 It may contain 2, 23, 24, 25, 26, 27, 28, or 29 amino acid substitutions. In some embodiments, sequence identity is determined by the antigen-binding domain that binds to hK2 of the Disclosure. Approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, and 88% of those percentages. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or it may be 99%. In some embodiments, diversity exists in the framework domain. It exists. In some embodiments, variants are generated by conservative substitution.

[0505] For example, the antigen-binding domain that binds to hK2 is located at residue positions D16 and A23 in VH. , P41, G45, I49, M70, and A88, and V89 (hu11B of sequence number 5) 6_Residue numbering according to VH) and D1, D9, S10, A12, V13, L in VL 15, I21, N22, K24, K49, R58, V62, D64, S80, L82, Q 83, A84, V87, A88, Y91, Q104, and L108 (hu1 of sequence number 2) Substitutions may be included in the residue numbering according to 1B6_VL. Conservative substitutions can be at any designated position. This can be done in this manner, and the variant of the antigen-binding domain that binds to the obtained hK2 is expressed in this The desired properties are tested using the assay described in the detailed manual.

[0506] In some embodiments, the isolated protein containing an antigen-binding domain that binds to hK2 is The antigen-binding domains VH and VL that bind to hK2 as disclosed herein are small in number. At least 80% (for example, at least 85%, at least 90%, at least 95%, or This includes VH and VL, which are at least 99% identical.

[0507] Furthermore, antigens that bind to hK2, including VH and VL which are at least 80% identical to the following. Joint domains are also provided. VH of sequence number 137 and VL of sequence number 138; VH of sequence number 139 and VL of sequence number 140; VH of sequence number 4 and VL of sequence number 1; VH of sequence number 4 and VL of sequence number 2; VH of sequence number 4 and VL of sequence number 3; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; VH of sequence number 6 and VL of sequence number 1; VH of sequence number 6 and VL of sequence number 2; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3; VH of sequence number 159 and VL of sequence number 160; VH of sequence number 159 and VL of sequence number 140; VH of sequence number 161 and VL of sequence number 140; VH of sequence number 162 and VL of sequence number 163; VH of sequence number 164 and VL of sequence number 165; VH of sequence number 166 and VL of sequence number 167; VH of sequence number 168 and VL of sequence number 169; or VH of sequence number 204 and VL of sequence number 205.

[0508] In some embodiments, the identity is 85%. The identity rate is 90%. In some embodiments, the identity rate is 91%. In terms of form, the identity is 91%. In some embodiments, the identity is 92%. In some embodiments, the identity is 93%. The percentage is 94%. In some embodiments, the percentage of identity is 94%. Morphologically, the identity is 95%. In some embodiments, the identity is 96%. In some embodiments, the identity is 97%. The accuracy rate is 98%. In some embodiments, the accuracy rate is 99%.

[0509] In some embodiments, the antigen-binding domain that binds to hK2 is V of SEQ ID NO: 137 H and at least 80% (for example, at least 85%, at least 90%, at least 95%) VH and VL of SEQ ID NO: 138 are identical in % or at least 99% of the original VH and at least 80% of the original VH. % (For example, at least 85%, at least 90%, at least 95%, or at least Includes VL which is 99% identical.

[0510] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. H and at least 80% (for example, at least 85%, at least 90%, at least 95%) VH and VL of SEQ ID NO: 163 are identical in % or at least 99% of the original VH and at least 80% of the original VH. % (For example, at least 85%, at least 90%, at least 95%, or at least Includes VL which is 99% identical.

[0511] In some embodiments, the antigen-binding domain that binds to hK2 is V of SEQ ID NO: 164 H and at least 80% (for example, at least 85%, at least 90%, at least 95%) VH and VL of Sequence ID No. 165 are identical in % or at least 99% of the original VH and at least 80% of the original VH. % (For example, at least 85%, at least 90%, at least 95%, or at least Includes VL which is 99% identical.

[0512] In some embodiments, the antigen-binding domain that binds to hK2 is V of SEQ ID NO: 166 H and at least 80% (for example, at least 85%, at least 90%, at least 95%) VH and VL of SEQ ID NO: 167 are identical in % or at least 99% of the original VH and at least 80% of the original VH. % (For example, at least 85%, at least 90%, at least 95%, or at least Includes VL which is 99% identical.

[0513] In some embodiments, the antigen-binding domain that binds to hK2 is V of SEQ ID NO: 166 H and at least 80% (for example, at least 85%, at least 90%, at least 95%) VH and VL of sequence number 444 are identical in % or at least 99% of the original VH and at least 80% of the original VH. % (For example, at least 85%, at least 90%, at least 95%, or at least Includes VL which is 99% identical.

[0514] In some embodiments, the antigen-binding domain that binds to hK2 is V of SEQ ID NO: 168 H and at least 80% (at least 85%, at least 90%, at least 95%, or VH is identical to VL of sequence number 169 by at least 99%, and VH is identical to VL of sequence number 169 by at least 80% (for example) (For example, at least 85%, at least 90%, at least 95%, or at least 99%) Includes VL which is identical.

[0515] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 204. H and at least 80% (at least 85%, at least 90%, at least 95%, or VH is identical to VL of sequence number 205 by at least 99%, and VH is identical to VL by at least 80% (for example) (For example, at least 85%, at least 90%, at least 95%, or at least 99%) Includes VL which is identical.

[0516] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It includes VH, which is at least 85% identical to H, and VL of sequence number 163.

[0517] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VH, which is at least 90% identical to H, and VL of sequence number 163.

[0518] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It includes VH, which is at least 91% identical to H, and VL of sequence number 163.

[0519] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It includes VH, which is at least 92% identical to H, and VL of sequence number 163.

[0520] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VH, which is at least 93% identical to H, and VL of sequence number 163.

[0521] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It includes VH, which is at least 94% identical to H, and VL of sequence number 163.

[0522] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VH, which is at least 95% identical to H, and VL of sequence number 163.

[0523] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It includes VH, which is at least 96% identical to H, and VL of sequence number 163.

[0524] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It includes VH, which is at least 97% identical to H, and VL of sequence number 163.

[0525] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It includes VH, which is at least 98% identical to H, and VL of sequence number 163.

[0526] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VH, which is at least 99% identical to H, and VL of sequence number 163.

[0527] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. It contains VL that is at least 85% identical to H and VL of Sequence ID No. 163.

[0528] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 90% identical to H and VL of Sequence ID No. 163.

[0529] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 91% identical to H and VL of Sequence ID No. 163.

[0530] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 92% identical to H and VL of Sequence ID No. 163.

[0531] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 93% identical to H and VL of Sequence ID No. 163.

[0532] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 94% identical to H and VL of Sequence ID No. 163.

[0533] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 95% identical to H and VL of Sequence ID No. 163.

[0534] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Contains VL that is at least 96% identical to H and VL of Sequence ID No. 163.

[0535] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 97% identical to H and VL of Sequence ID No. 163.

[0536] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 98% identical to H and VL of Sequence ID No. 163.

[0537] In some embodiments, the antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. Includes VL that is at least 99% identical to H and VL of Sequence ID No. 163.

[0538] The identity percentage between the two sequences is introduced for optimal alignment of the two sequences. The same shared by the array, taking into account the number of gaps that need to be filled and the length of each gap. It is a function of the number of positions (i.e., identity % = number of identical positions / total number of positions × 100).

[0539] The percentage of identity between two amino acid sequences is calculated using the ALIGN program (version 2). 0) The algorithms of E. Meyers and W. Miller incorporated into (Com Using put.Appl.Biosci 4:11-17(1988)), PAM12 Calculated using a weighted residue table, gap length penalty 12, and gap penalty 4. It can be determined that the percentage of identity between two amino acids or nucleic acid sequences is GCG. The software package (available at http: / / www.gcg.com) is now generally available. The Needleman and Wunsch algorithms incorporated into the P program ( Using J.Mol.Biol.48:444-453(1970), Blossom 62 matrix or PAM250 matrix, and gap weightings 16, 14, 12 , 10, 8, 6, or 4 and length weighting 1, 2, 3, 4, 5, or 6 are used to determine It is also possible to do so.

[0540] In some embodiments, the variant of the antigen-binding domain that binds to hK2 is hK2 While retaining the desired functional properties of the parent antigen-binding fragment that binds to one of the CDR regions Includes one or two conservative substitutions.

[0541] "Conservative modifications" can have a significant impact on the binding properties of antibodies, including those involving amino acid modifications. This refers to amino acid modifications that do not alter the amino acid. Conservative modifications include amino acid substitution, addition, and Includes deletions. Conservative amino acid substitutions are when an amino acid is replaced by an amino acid residue with a similar side chain. This is a substitution that replaces [the original amino acid]. A family of amino acid residues with similar side chains is clearly [the original amino acid]. Defined as having acidic side chains (e.g., aspartic acid, glutamic acid) and basic side chains (e.g., For example, lysine, arginine, histidine), nonpolar side chains (for example, alanine, valine, ro (Isine, isoleucine, proline, phenylalanine, methionine), non-charged side chains ( For example, glycine, asparagine, glutamine, cysteine, serine, threonine, tyrofoam Syn, tryptophan), aromatic side chain (e.g., phenylalanine, tryptophan, hyphenalanine) Stidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, Isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), β-branched side chains (e.g., threonine, valine, isoleucine), and sulfur-containing side chains (cystay It contains amino acids (such as methionine). Furthermore, regarding alanine scanning mutagenesis... As previously explained (MacLennan et al., (1988)Ac ta Physiol Scand Suppl 643:55-67, Sasaki et al., (1988) Adv Biophys 35:1-24), polypeptide Any of the natural residues within can be substituted with alanine. The amino acid substitutions for the antibody of the present invention are This can be done by known methods, such as PCR mutagenesis (U.S. Patent No. 4,683,195). It is possible to do so. Alternatively, the variant library could be, for example, a random codon (NN). K) or non-random codons (e.g., 11 amino acids (Ala, Cys, Asp, Glu) DVK codes that code Gly, Lys, Asn, Arg, Ser, Tyr, Trp It can also be generated using (n). The resulting variant can be used in the assay described herein. Its characteristics can be tested using this method.

[0542] Method for producing antigen-binding fragments that bind to hK2 The antigen-binding domains that bind to hK2 provided in this disclosure were prepared using various techniques. This can be done. For example, by using the Kohler-Milstein hybridoma method. This method allows for the identification of VH / VL pairs that bind to hK2. , mice or other host animals, such as hamsters, rats, or chickens, and humans and Immunize with cynomolgus monkey hK2, followed by immunization with myeloma cells using standard methods. The spleen cells of the animal are fused to form hybridoma cells. Colonies derived from ibridomas are evaluated based on binding specificity, cross-reactivity, or lack thereof, and antigenicity. Antigens that bind to hK2 having desired properties such as affinity and any desired functionality. Screening may be performed for the production of antibodies containing a binding domain.

[0543] The antigen-binding domain that binds to hK2 produced by immunizing non-human animals It may be humanized. Examples of humanization techniques include the selection of a human acceptor framework. This includes CDR grafting (US Patent No. 5,225,539) and SDR grafting (US Patent No. 5,225,539). License No. 6,818,749), Resurfacing (Padlan, (1991) Mol Immunol 28:489-499), resurfacing of specificity-determining residues (US specific (Publication No. 2010 / 0261620), Human Framework Adaptation (U.S. 8, Examples include patents 748,356, or hyperhumanization (U.S. Patent No. 7,709,226). These methods determine the similarity of CDR lengths or the identity of their canonical structures, or these Based on the combination, it can be selected based on its overall homology to the parent framework. A human framework is transplanted with the CDR or a subset of the CDR residues of the parent antibody.

[0544] The humanized antigen-binding domain is described in International Publication No. 1090 / 007861 and International Publication No. 1992 / The framework support structure has been modified using technologies such as those described in Patent No. 22653. Either by incorporating a group to maintain binding affinity (reverse mutation), or by CDR. By introducing diversity, for example, by improving the affinity of the antigen-binding domain, the desired It may be further optimized to improve its selectivity or affinity for the antigen.

[0545] Mice, rats, or Using transgenic animals such as chickens, antigen-binding fragments that bind to hK2 are created. These can be manufactured, for example, under U.S. Patent No. 6,150,584, International Publication No. 19 99 / 45962, 2002 / 066630, 2002 / 43478, 2002 / 066630, 2002 / 43478, This is described in issues 2002 / 043478 and 1990 / 04036. The endogenous immunoglobulin gene locus of such animals may be disrupted or deleted, homologous or non- Using homologous recombination, using transchromosomes, or minigenes Using this method, at least one complete or partial human immunoglobulin locus can be identified in animals. It can be inserted into the genome. Regeneron (http: / / _www_regene ron_com), Harbor Antibodies(http: / / _www_ harborantibodies_com), Open Monoclonal T echnology, Inc.(OMT)(http: / / _www_omtinc_n et), KyMab (http: / / _www_omtinc_net), Triann i(http: / / _www.trianni_com), and Ablexis(htt Companies such as p: / / _www_ablexis_com use the above technology to select There are efforts underway to provide human antibodies that target specific antigens. Several implementations Morphologically, Ablexis mice and OmniRat rats were given soluble full-length KLK2 tannins. Immunized with protein (human kallikrein-2 6-His protein) (SEQ ID NO: 454) .

[0546] The antigen-binding domain that binds to hK2 is human immunoglobulin or a part thereof, for example, Fa b. A single-chain antibody (scFv), or a variable region of an unpaired or synpaired antibody, is expressed. The phage can be selected from a phage display library that has been genetically modified. The antigen-binding domain that binds to hK2 is, for example, Shi et al., (2010). (J Mol Biol 397:385-96 and International Publication No. 09 / 085462) As a fusion protein with the bacteriophage pIX coat protein described, it is an antimicrobial agent. Isolation from a phage display library expressing the body's heavy chain and light chain variable regions. This is possible. Regarding the library's ability to bind phages to human and / or cynomolgus monkey hK2. Screening is performed, and the obtained positive clones are further characterized, and from the clone lysates... Fab may be isolated and converted into scFV or other components of the antigen-binding fragment.

[0547] Preparation of immunogenic antigens and expression and generation of the antigen-binding domains of this disclosure are recombinant tangents. This can be carried out using any suitable technique, such as protein synthesis. Immunogenic antigens are sperm Protein preparation, or protein mixtures containing whole cells or cell or tissue extracts. It may be administered to animals in this manner, or the antigen may encode the antigen or a part thereof. They may be newly formed (de novo) within the animal's body from existing nucleic acids.

[0548] Conjugation to the half-life extension portion The antigen-binding domain that binds to hK2 in this disclosure is conjugated to the half-life extension portion. . exemplified half-life extension portions include albumin, albumin variants, and albumin. Binding proteins and / or domains, transferrin and its fragments and analogs, immuno Globulin (Ig) or a fragment thereof, for example, the Fc region. The aforementioned half-life extension portion The no-acid sequence is publicly known. Ig or its fragments are all isotypes, i.e., IgG1 It includes IgG2, IgG3, IgG4, IgM, IgA, and IgE.

[0549] Additional half-life extension that may be conjugated to the antigen-binding domain that binds to hK2 in this disclosure The long portion is polyethylene glycol (PEG) molecule for the desired properties, for example PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, e.g. For example, laurate ester, myristic acid ester, stearate ester, arachidin Acid esters, behenate esters, oleate esters, arachidonic acid esters, octane Dioxides, tetradecane dioxide, octadecane dioxide, docosanedioic acid, polylysine, octane These include carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides). The portion may be directly fused with the antigen-binding domain that binds to hK2 of the present disclosure, and a standard cross They may be prepared by conditioning and expression techniques, or by well-known chemical coupling methods. Using this method, the antigen-binding domain that binds to the recombinant hK2 of this disclosure is modified. You may combine them.

[0550] For example, incorporating a cysteine ​​residue into the C-terminus of the antigen-binding domain that binds to hK2 of this disclosure. Either insert or genetically modify the cysteine ​​residues so that they face away from the hK2 binding site. By placing the pegyl group in place and bonding it to cysteine ​​using a well-known method, the present disclosure The pegyl portion can be conjugated to the antigen-binding domain that binds to hK2.

[0551] In some embodiments, the antigen-binding fragment that binds to hK2 is condiment in the half-life extension portion. It will be gated.

[0552] In some embodiments, the half-life extension portion is immunoglobulin (Ig), Ig fragments, Ig constant region, fragment of Ig constant region, Fc region, transferrin, albumin, albumin The mine-binding domain is or polyethylene glycol. In some embodiments, half The prolonged period is the steady-state Ig region.

[0553] In some embodiments, the half-life extension portion is Ig.

[0554] In some embodiments, the half-life extension portion is a fragment of Ig.

[0555] In some embodiments, the half-life extension portion is the Ig steady-state region.

[0556] In some embodiments, the half-life extension portion is a fragment of the Ig steady-state region.

[0557] In some embodiments, the half-life extension portion is the Fc region.

[0558] In some embodiments, the half-life extension portion is albumin.

[0559] In some embodiments, the half-life extension portion is the albumin-binding domain.

[0560] In some embodiments, the half-life extension portion is transferrin.

[0561] In some embodiments, the half-life extension portion is polyethylene glycol.

[0562] The antigen-binding domain that binds to hK2 conjugated to the half-life extension portion is a known The pharmacokinetic properties can be evaluated using the vivo model.

[0563] Conjugation to the constant region of immunoglobulin (Ig) or fragments of the constant region of Ig The antigen-binding domain that binds to hK2 in this disclosure is either the Ig constant region or a fragment of the Ig constant region. It conjugates to Fc effector function C1q binding, complement-dependent cell damage (CDC). ), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, or cell surface Antibody-like properties including downregulation of surface receptors (e.g., B cell receptors; BCRs) It is possible to impart properties to the Ig steady-state region or fragments of the Ig steady-state region. As such, it also functions as a half-life extension portion. Antigen binding to hK2 of the present disclosure The domain can be genetically engineered using standard methods to create a conventional full-length antibody. Full-length antibodies containing an antigen-binding domain that binds to hK2 are further described herein. It may be genetically modified.

[0564] The constant region of the immunoglobulin heavy chain consists of subdomains CH1, hinge, CH2, and CH3. It is composed of residues numbered according to the EU index, with the CH1 domain being residue A in the heavy chain. The CH2 domain residues extend from 118 to V215, and the CH3 domain residues extend from A231 to K340. It extends to residues G341-K447. In some cases, G341 is the CH2 domain residue. It is called the base. The hinge generally contains E216 and terminates at P230 of human IgG1. It is defined as follows: The Ig Fc region consists of at least the CH2 and CH3 domains of the Ig steady state region. This includes, and therefore, the Ig heavy chain constant region from at least approximately A231 to K447. Includes the region.

[0565] The present invention also provides a conjugate to the immunoglobulin (Ig) constant region or a fragment of the Ig constant region. It provides an antigen-binding domain that binds to the modified hK2.

[0566] In some embodiments, the Ig steady-state region is the heavy chain steady-state region.

[0567] In some embodiments, the Ig steady-state region is the light chain steady-state region.

[0568] In some embodiments, the Ig steady-state region fragment includes an Fc region.

[0569] In some embodiments, the Ig constant region fragment includes a CH2 domain.

[0570] In some embodiments, the Ig constant region fragment includes a CH3 domain.

[0571] In some embodiments, the Ig steady-state region fragment consists of the CH2 domain and the CH3 domain. Includes.

[0572] In some embodiments, the Ig steady-state region fragment is at least part of the hinge, CH2 It includes the main and CH3 domains. Part of the hinge consists of one or more amino acids of the Ig hinge. It refers to a residue.

[0573] In some embodiments, the Ig steady-state region fragment comprises a hinge, a CH2 domain, and a CH2 domain. Includes 3 domains.

[0574] In some embodiments, the antigen-binding domain that binds to hK2 is the Ig constant region or I It is conjugated to the N-terminus of the g-stationary region fragment.

[0575] In some embodiments, the antigen-binding domain that binds to hK2 is the Ig constant region or I It is conjugated to the C-terminus of the g-stationary region fragment.

[0576] In some embodiments, the antigen-binding domain that binds to hK2 is a second linker (L 2) Conjugate to the Ig steady-state region or a fragment of the Ig steady-state region.

[0577] In some embodiments, L2 is represented by sequence numbers 7, 76, 77, 78, 79, 80, 81. , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 1 It contains the amino acid sequence 06, 107, or 108.

[0578] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 7.

[0579] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 76.

[0580] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 77.

[0581] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 78.

[0582] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 79.

[0583] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 80.

[0584] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 81.

[0585] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 82.

[0586] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 83.

[0587] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 84.

[0588] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 85.

[0589] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 86.

[0590] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 87.

[0591] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 88.

[0592] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 89.

[0593] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 90.

[0594] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 91.

[0595] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 92.

[0596] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 93.

[0597] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 94.

[0598] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 95.

[0599] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 96.

[0600] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 97.

[0601] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 98.

[0602] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 99.

[0603] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 100.

[0604] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 101.

[0605] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 102.

[0606] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 103.

[0607] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 104.

[0608] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 105.

[0609] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 106.

[0610] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 107.

[0611] In some embodiments, L2 includes the amino acid sequence of SEQ ID NO: 108.

[0612] The hK2 of this disclosure is bound to the Ig constant region or a fragment of the Ig constant region. The antigen-binding domain will be evaluated for its functionality using several known assays. The binding to hK2 can be evaluated using the method described herein. Changes induced by the Ig steady domain or fragments of the Ig steady region, such as the Fc region. The characteristics include FcγRI, FcγRII, FcγRIII, or FcRn receptors. In Fc receptor binding assays using the soluble form of the receptor, or, for example, ADCC Assay using a cell-based assay that measures CDC or ADCP. It is possible.

[0613] ADCC targets hK2-expressing cells as target cells and NK cells as effector cells. It can be evaluated using an in vitro assay. Cell lysis is performed by lysing the cells. Release of labels from cells (e.g., radioactive substrates, fluorescent dyes, or native intracellular proteins) It can be detected by the following. In an exemplary assay, target cells are compared to target cell 1. The effector cells are used in a ratio of 4. Target cells are pre-labeled with BATDA, and the effector cells are used. Combine with cells and test antibody. Incubate the sample for 2 hours and release into the supernatant. Cell lysis was measured by measuring BATDA. 0.67% Triton X Data normalized to the maximum cytotoxicity caused by -100 (Sigma Aldrich). Furthermore, the spontaneous release of BATDA from target cells in the absence of any antibody is the most effective method. Find a small contrast.

[0614] ADCP uses monocyte-derived macrophages as effector cells, and GFP or Any hK2-expressing cell genetically engineered to express other labeled molecules can be used as target cells. It can be evaluated by using it. In an example assay, effector: label The target cell ratio can be, for example, 4:1. Effector cells are treated with the antibody of the present invention. Incubation may be performed with target cells for 4 hours, either with or without addition. Afterward, the cells can be detached using actase. Macrophages are bound to the fluorescent label. It can be identified by anti-CD11b antibodies and anti-CD14 antibodies, and the phagocytic percentage is standard Using this method, CD11 + and CD14 + Percentage of GFP fluorescence in macrophages ( It can be determined based on the percentage.

[0615] The CDC of cells is, for example, Doudi cells RPMI-B (RP supplemented with 1% BSA). MI) 1 x 10 5 Plate with cells / well (50 μL / well), then add 50 μL Add the test protein to the wells at a final concentration of 0-100 μg / mL, and stir the reaction mixture at room temperature for 1 Incubate for 5 minutes, add 11 μL of pooled human serum to the well, and mix the reaction mixture for 3 minutes. The measurement can be performed by incubating the lysed cells at 7°C for 45 minutes. Percentages (%) are given using the standard method for propidium iodide staining in FACS assays. It can be detected as a percentage of cells.

[0616] In some embodiments, the antigen-binding domain that binds to hK2 is located in the IgG1 heavy chain constant region. It is conjugated to a region or a fragment of the IgG1 heavy chain constant region. In some embodiments, The antigen-binding domain that binds to hK2 is at least 80% (for example, less) of the sequence number 110. (At least 85%, at least 90%, at least 95%, or at least 99%) identical A constant region of IgG monohelic acid containing a certain amino acid sequence, or a fragment of a constant region of IgG monohelic acid (for example) , conjugate to the hinge-CH2-CH3. In some embodiments, The IgG1 heavy chain constant region is affected by Fc silencing mutations (L234A_L235A_D265 S) and T350V_T366L_K designed to promote selective heterodimerization Includes the 392L_T394W mutation. In some embodiments, antigen binding to hK2. The domain is the IgG1 heavy chain constant region having the amino acid sequence of SEQ ID NO: 110 or IgG1 It is conjugated into fragments of the heavy chain constant region.

[0617] In some embodiments, the antigen-binding domain that binds to hK2 is the A of Sequence ID No. 378 Mino acid sequence and at least 80% (e.g., at least 85%, at least 90%, less) The IgG1 heavy chain constant region containing amino acid sequences that are 95% or at least 99% identical It is conjugated into a region (e.g., CH1-hinge-CH2-CH3). Specific implementation In this state, the antigen-binding domain that binds to hK2 contains the amino acid sequence of SEQ ID NO: 378. It is conjugated to a fragment of the gG1 heavy chain constant region or the IgG1 heavy chain constant region.

[0618] In some embodiments, the antigen-binding domain that binds to hK2 is less than the one specified by SEQ ID NO: 309. At least 80% (for example, at least 85%, at least 90%, at least 95%, or The IgG1 light chain constant region contains an amino acid sequence that is at least 99% identical to that of a conjugate. In some embodiments, the antigen-binding domain that binds to hK2 is the sequence number. 447 and at least 80% (for example, at least 85%, at least 90%, at least Conjugated to the IgG1 light chain constant region which is 95% or at least 99% identical. In some embodiments, the antigen-binding domain that binds to hK2 is sequence number 309 or It is conjugated into the constant region of the IgG1 light chain, which contains the same amino acid sequence as 447. In certain embodiments, the antigen-binding domain that binds to hK2 is the same as that of SEQ ID NO: 309. It is conjugated into the constant region of the IgG1 light chain, which contains a certain amino acid sequence.

[0619] In certain embodiments, the isolated protein disclosed herein is the amine of SEQ ID NO: 378. An antigen-binding molecule that binds to hK2 is conjugated to the constant region of the IgG1 heavy chain containing an acid sequence. The constant region of the IgG1 light chain contains the same amino acid sequence as the main and SEQ ID NO: 309. It contains an antigen-binding domain that binds to jugated hK2.

[0620] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain and the first Ig weight containing the same amino acid sequence as SEQ ID NO: 378 It includes a chain-steady region or a fragment of the first Ig heavy chain-steady region.

[0621] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain and the first Ig cell containing the same amino acid sequence as SEQ ID NO: 309 It includes a chain steady region or a fragment of the first Ig light chain steady region.

[0622] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. (i) The antigen-binding domain and the first I containing the same amino acid sequence as SEQ ID NO: 378 (ii) a fragment of the g heavy chain steady region or the first Ig heavy chain steady region, and the same as SEQ ID NO: 309 A first Ig light chain constant region or a fragment of the first Ig light chain constant region containing a certain amino acid sequence include.

[0623] In certain embodiments, the isolated proteins disclosed herein are lymphocyte antigens (e.g., A second antigen-binding domain that binds to CD3, and an amino acid identical to that of Sequence ID No. 109. It contains a second Ig constant region containing an acid sequence or a fragment of the second Ig constant region.

[0624] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. (i) The antigen-binding domain and the first I containing the same amino acid sequence as SEQ ID NO: 378 (ii) a fragment of the g heavy chain steady region or the first Ig heavy chain steady region, and the same as SEQ ID NO: 309 A first Ig light chain constant region or a fragment of the first Ig light chain constant region containing a certain amino acid sequence The isolated protein contains a second antigenic binding to lymphocyte antigens (e.g., CD3). A synthetic domain and a second Ig constant region containing the same amino acid sequence as SEQ ID NO: 109 or It further includes a fragment of the second Ig steady-state region.

[0625] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and The first antigen-binding domain that binds to hK2, including LCDR3, is identical to SEQ ID NO: 378. A first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region containing the amino acid sequence Includes.

[0626] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and The first antigen-binding domain that binds to hK2, including LCDR3, is identical to SEQ ID NO: 309. A first Ig light chain constant region or a fragment of the first Ig light chain constant region containing the amino acid sequence and Includes.

[0627] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and (i)SEQ ID NO: 378 A first Ig heavy chain constant region containing an amino acid sequence identical to that of the first Ig heavy chain constant region (ii) the fragment and the first Ig light chain constant region containing the same amino acid sequence as SEQ ID NO: 309 It includes a region or a fragment of the first Ig light chain constant region.

[0628] In certain embodiments, the isolated proteins disclosed herein are lymphocyte antigens (e.g., A second antigen-binding domain that binds to CD3, and is the HCDR1 of sequence number 255. HCDR2 (sequence number 256), HCDR3 (sequence number 257), LCDR (sequence number 258) 1. Lymphocyte anti- It contains a second antigen-binding domain that binds to the source, and an amino acid sequence identical to that of sequence number 109. It includes a second Ig steady-state region or a fragment of the second Ig steady-state region.

[0629] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and (i)SEQ ID NO: 378 A first Ig heavy chain constant region containing an amino acid sequence identical to that of the first Ig heavy chain constant region (ii) the fragment and the first Ig light chain constant region containing the same amino acid sequence as SEQ ID NO: 309 The isolated protein comprises a region or a fragment of the first Ig light chain constant region, and the isolated protein is a lymphocyte antigen ( For example, a second antigen-binding domain that binds to CD3, and the HCD of sequence number 255. R1, HCDR2 (sequence number 256), HCDR3 (sequence number 257), L (sequence number 258) Including CDR1, LCDR2 of sequence number 259, and LCDR3 of sequence number 261, A second antigen-binding domain that binds to the Pacyl antigen, and an amino acid sequence identical to that of SEQ ID NO: 109. Further comprising a second Ig steady-state region or a fragment of the second Ig steady-state region containing a column.

[0630] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and the amino acid sequence identical to that of SEQ ID NO: 378. It includes a first Ig heavy chain steady region or a fragment of the first Ig heavy chain steady region.

[0631] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and the amino acid sequence identical to that of SEQ ID NO: 309. It comprises a first Ig light chain constant region or a fragment of the first Ig light chain constant region.

[0632] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and (i) an amino acid identical to that of Sequence ID No. 378. (ii) a first Ig heavy chain constant region containing an acid sequence or a fragment of the first Ig heavy chain constant region, and The first Ig light chain constant region or the first Ig light chain containing the same amino acid sequence as column number 309 It includes fragments of the chain-steady region.

[0633] In certain embodiments, the isolated proteins disclosed herein are lymphocyte antigens (e.g., A second antigen-binding domain that binds to CD3, and the amino acid sequence of SEQ ID NO: 331 It includes a second antigen-binding domain that binds to lymphocyte antigens, and is identical to SEQ ID NO: 109. It contains a second Ig constant region or a fragment of a second Ig constant region that includes a certain amino acid sequence.

[0634] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and (i) an amino acid identical to that of Sequence ID No. 378. (ii) a first Ig heavy chain constant region containing an acid sequence or a fragment of the first Ig heavy chain constant region, and The first Ig light chain constant region or the first Ig light chain containing the same amino acid sequence as column number 309 The isolated protein comprises a fragment of the chain constant region, and the isolated protein is a lymphocyte antigen (e.g., CD3). A second antigen-binding domain that binds, comprising the amino acid sequence of SEQ ID NO: 331, A second antigen-binding domain that binds to the Pacyl antigen, and an amino acid sequence identical to that of SEQ ID NO: 109. Further comprising a second Ig steady-state region or a fragment of the second Ig steady-state region containing a column.

[0635] Proteins containing an antigen-binding domain that binds to hK2 (as disclosed herein) The antigen-binding domain that binds to hK2 in this disclosure can be designed in various ways using standard methods. They can be genetically engineered to produce monospecific or multispecific proteins.

[0636] This disclosure also includes a single-specificity antigen-binding domain that binds to the hK2 of this disclosure. We provide protein.

[0637] In some embodiments, the single-specific protein is an antibody.

[0638] This disclosure also relates to a multispecific protein containing an antigen-binding domain that binds to hK2. We provide quality.

[0639] In some embodiments, the multispecific protein is bispecific.

[0640] In some embodiments, the multispecific protein is triple specific.

[0641] In some embodiments, the multispecific protein is quadruplespecific.

[0642] In some embodiments, the multispecific protein is monovalent with respect to binding to hK2. ru.

[0643] In some embodiments, the multispecific protein is divalent for binding to hK2. ru.

[0644] This disclosure also relates to a first antigen-binding domain that binds to hK2 and a lymphocyte antigen (CD3) This provides an isolated, multispecific protein containing a second antigen-binding domain that binds to ( ).

[0645] In some embodiments, the lymphocyte antigen is a T cell antigen.

[0646] In some embodiments, the T cell antigen is CD8 + It is a T cell antigen.

[0647] In some embodiments, the lymphocyte antigen is the NK cell antigen.

[0648] In some embodiments, the lymphocyte antigens are CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, These are BTNL8, PD-1, CD195, or NKG2C.

[0649] In some embodiments, the lymphocyte antigen is CD3ε.

[0650] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0651] In some embodiments, the anti-hK2 / anti-CD3 protein exhibits bispecificity.

[0652] In some embodiments, a first antigen-binding domain and / or lymphatic domain that binds to hK2. The second antigen-binding domain that binds to the globular antigen is scFv, (scFv)2, Fv, Fab Includes F(ab')2, Fd, dAb, or VHH.

[0653] In some embodiments, a first antigen-binding domain and / or lymphatic domain that binds to hK2. The second antigen-binding domain, which binds to the globular antigen, contains Fab.

[0654] In certain embodiments, the first antigen-binding domain that binds to hK2 includes Fab.

[0655] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is Fab Includes.

[0656] In some embodiments, a first antigen-binding domain and / or lymphatic domain that binds to hK2. The second antigen-binding domain that binds to the globular antigen contains F(ab')2.

[0657] In some embodiments, the first antigen-binding domain that binds to hK2 is F(ab') Includes 2.

[0658] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is F(a Includes b')2

[0659] In some embodiments, a first antigen-binding domain and / or lymphatic domain that binds to hK2. The second antigen-binding domain, which binds to the globular antigen, contains VHH.

[0660] In some embodiments, the first antigen-binding domain that binds to hK2 includes VHH. .

[0661] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is VHH Includes.

[0662] In some embodiments, a first antigen-binding domain and / or lymphatic domain that binds to hK2. The second antigen-binding domain, which binds to the globular antigen, contains Fv.

[0663] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fv.

[0664] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is Fv include.

[0665] In some embodiments, a first antigen-binding domain and / or lymphatic domain that binds to hK2. The second antigen-binding domain, which binds to the globular antigen, contains Fd.

[0666] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fd.

[0667] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is Fd include.

[0668] In some embodiments, a first antigen-binding domain and / or lymphatic domain that binds to hK2. The second antigen-binding domain, which binds to the globular antigen, contains scFV.

[0669] In certain embodiments, the multispecific protein is a first antigen-binding domain that binds to hK2. The fa contains a second antigen-binding domain that binds to lymphocyte antigens (e.g., CD3). n is a bispecific compound that includes scFv.

[0670] In some embodiments, the first antigen-binding domain to bind includes an scFV.

[0671] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is scF Includes V

[0672] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV. Furthermore, the second antigen-binding domain that binds to lymphocyte antigens contains Fab.

[0673] In certain embodiments, the first antigen-binding domain that binds to hK2 includes Fab, The second antigen-binding domain, which binds to the lymphocyte antigen, contains scFV.

[0674] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV. Furthermore, the second antigen-binding domain that binds to lymphocyte antigens contains Fab'.

[0675] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fab'. Furthermore, the second antigen-binding domain that binds to lymphocyte antigens contains scFV.

[0676] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV. Furthermore, the second antigen-binding domain that binds to lymphocyte antigens contains Fv.

[0677] In some embodiments, the first antigen-binding domain that binds to hK2 includes dAb. The second antigen-binding domain, which binds to lymphocyte antigens, contains scFV.

[0678] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV. Furthermore, the second antigen-binding domain that binds to lymphocyte antigens contains dAb.

[0679] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fd, The second antigen-binding domain, which binds to lymphocyte antigens, contains scFV.

[0680] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV. Furthermore, the second antigen-binding domain that binds to lymphocyte antigens contains VHH.

[0681] In some embodiments, the first antigen-binding domain that binds to hK2 includes VHH. The second antigen-binding domain, which binds to lymphocyte antigens, contains scFV.

[0682] In some embodiments, the first antigen-binding domain that binds to hK2 includes Fv, The second antigen-binding domain, which binds to lymphocyte antigens, contains scFV.

[0683] In some embodiments, the first antigen-binding domain that binds to hK2 includes scFV. Furthermore, the second antigen-binding domain that binds to lymphocyte antigens contains Fd.

[0684] In some embodiments, scFv is VH, first ri, from the N-terminus to the C-terminus. Inker (L1), and VL (VH-L1-VL), or VL, L1, and VH (VL-L Includes 1-VH).

[0685] In some embodiments, L1 contains approximately 5 to 50 amino acids.

[0686] In some embodiments, L1 contains approximately 5 to 40 amino acids.

[0687] In some embodiments, L1 contains approximately 10 to 30 amino acids.

[0688] In some embodiments, L1 contains approximately 10 to 20 amino acids.

[0689] In some embodiments, L1 is represented by sequence numbers 7, 76, 77, 78, 79, 80, 81. , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 1 It contains the amino acid sequence 06, 107, or 108.

[0690] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 7.

[0691] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 76.

[0692] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 77.

[0693] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 78.

[0694] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 79.

[0695] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 80.

[0696] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 81.

[0697] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 82.

[0698] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 83.

[0699] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 84.

[0700] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 85.

[0701] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 86.

[0702] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 87.

[0703] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 88.

[0704] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 89.

[0705] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 90.

[0706] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 91.

[0707] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 92.

[0708] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 93.

[0709] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 94.

[0710] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 95.

[0711] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 96.

[0712] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 97.

[0713] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 98.

[0714] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 99.

[0715] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 100.

[0716] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 101.

[0717] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 102.

[0718] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 103.

[0719] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 104.

[0720] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 105.

[0721] In some embodiments, L1 comprises the amino acid sequence of SEQ ID NO: 106.

[0722] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 107.

[0723] In some embodiments, L1 includes the amino acid sequence of SEQ ID NO: 108.

[0724] In certain embodiments, L1 includes the amino acid sequence of SEQ ID NO: 7.

[0725] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 63. ,72,141,147,170,176,188,194,196,198,200, HCDR1 206 or 216, SEQ ID NOs: 64, 65, 73, 142, 148, 171 , 177, 188, 189, 195, 197, 199, 201, 207, or 217 H CDR2, Sequence IDs 66, 143, 172, 178, 184, 190, 208, or 21 HCDR3 of 8, SEQ ID NOs: 67, 68, 144, 173, 179, 182, 185, or LCDR1 191, SEQ ID NOs: 69, 70, 145, 174, 180, 186, 192, Or LCDR2 of 470, and sequence numbers 71, 146, 175, 181, 187, 193 , or including 209 LCDR3.

[0726] In some embodiments, the first antigen-binding domain that binds to hK2 is the following HCD Includes R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 Sequence IDs 63, 65, 66, 68, 70, and 71, respectively; Sequence numbers 63, 64, 66, 67, 69, and 71, respectively; Sequence IDs 63, 65, 66, 67, 69, and 71, respectively; Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; Sequence numbers 170, 171, 172, 173, 174, and 175, respectively; Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; Sequence numbers 170, 183, 184, 185, 186, and 187, respectively; Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; Sequence IDs 206, 207, 208, 182, 470, and 209, respectively; Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; Sequence numbers 72, 73, 66, 68, 70, and 71, respectively; Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; Sequence numbers 196, 197, 178, 179, 190, and 181, respectively; Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

[0727] In certain embodiments, the first antigen-binding domain that binds to hK2 is, respectively, SEQ ID NO: 170, 171, 172, 173, 174, and 175, or each corresponding to sequence number 194, HCDR1, HCDR2, HCDR3 of 195, 172, 173, 174, and 175 Includes LCDR1, LCDR2, and LCDR3.

[0728] In certain embodiments, the first antigen-binding domain that binds to hK2 is, respectively, SEQ ID NO: HCDR1, HCDR2, HC 170, 171, 172, 173, 174, and 175 Includes DR3, LCDR1, LCDR2, and LCDR3, and optionally CD3, CD3 Epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example. The second antigen-binding domain binds to the CD3 lymphocyte antigen.

[0729] In certain embodiments, the first antigen-binding domain that binds to hK2 is, respectively, SEQ ID NO: HCDR1, HCDR2, HC 194, 195, 172, 173, 174, and 175 Includes DR3, LCDR1, LCDR2, and LCDR3, and optionally CD3, CD3 Epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or NKG2C, for example. The second antigen-binding domain binds to the CD3 lymphocyte antigen.

[0730] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 13. Includes VH of 7 and VL of sequence number 138.

[0731] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. Includes VH of 2 and VL of sequence number 163.

[0732] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. Includes VH of 4 and VL of sequence number 165.

[0733] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. Includes VH of 6 and VL of sequence number 167.

[0734] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. Includes VH of 8 and VL of sequence number 169.

[0735] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 20 Includes VH of 4 and VL of SEQ ID NO: 205.

[0736] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 75 Includes VH and VL of sequence number 74.

[0737] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 75 Includes VH and VL of sequence number 74.

[0738] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. 2 VH and at least 80% (at least 85%, at least 90%, at least 95%) , or at least 99%) identical to VH and VL of SEQ ID NO: 163 and at least 80% (For example, at least 85%, at least 90%, at least 95%, or at least 9 9%) Includes VL which is identical.

[0739] In some embodiments, the first antigen-binding domain that binds to hK2 is, for example, a sequence Number 162 VH and at least 80% (e.g., at least 85%, at least 90%) Contains VH and VL of Sequence ID No. 163 that are at least 95% or at least 99% identical. nothing.

[0740] In some embodiments, the first antigen-binding domain that binds to hK2 is, for example, a sequence VH of number 162 and VL of sequence number 163 and at least 80% (for example, at least 8 Includes VL that is 5%, at least 90%, at least 95%, or at least 99% identical. nothing.

[0741] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. 2 VH and at least 80% (at least 85%, at least 90%, at least 95%) , or at least 99%) identical to VH and VL of SEQ ID NO: 163 and at least 80% (For example, at least 85%, at least 90%, at least 95%, or at least 9 9%) Includes the same VL, and optionally includes CD3, CD3 epsilon (CD3ε), and C D8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, B Lymphocyte antigens such as TNL8, PD-1, CD195, or NKG2C, for example, CD3 The second antigen-binding domain that binds.

[0742] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. 2 VH and at least 80% (at least 85%, at least 90%, at least 95%) , or including VH and VL of sequence number 163 which are identical (at least 99%), optionally CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2 D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195, or N KG2C is a second antigen-binding domain that binds to lymphocyte antigens, such as CD3.

[0743] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. VH of 2 and VL of SEQ ID NO: 163 and at least 80% (e.g., at least 85%, less Includes VLs that are identical by at least 90%, at least 95%, or at least 99%, Select CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1, CD195 , or a second antigen-binding domain that binds to lymphocyte antigens such as NKG2C, for example, CD3. .

[0744] In certain embodiments, the first antigen-binding domain that binds to hK2 is the one in Sequence ID No. 162. Includes VH and VL of sequence number 163.

[0745] In certain embodiments, the first antigen-binding domain that binds to hK2 is the one in Sequence ID No. 162. Includes VH and VL of sequence number 163, and optionally CD3, CD3 epsilon (CD3 ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD1 Lymphocytes such as 86, BTNL8, PD-1, CD195, or NKG2C, for example, CD3 A second antigen-binding domain that binds to the globular antigen.

[0746] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 4. VH of 5, 6, 139, 159, or 161 and SEQ ID NOs: 1, 2, 3, 140, or 16 Includes VL of 0, however Fab includes both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2 There's no need to worry.

[0747] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 4. It includes VH of 5 or 6 and VL of SEQ ID NO: 1, 2, or 3, provided that it is bound to hK2. The first antigen-binding domain includes both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2 do not have.

[0748] In some embodiments, the first antigen-binding domain that binds to hK2 includes: VH of sequence number 4 and VL of sequence number 1; VH of sequence number 4 and VL of sequence number 2; VH of sequence number 4 and VL of sequence number 3; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 4 and VL of SEQ ID NO: 160; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 1; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; VH of sequence number 6 and VL of sequence number 1; VH of sequence number 6 and VL of sequence number 2; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 3; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 140; VH of SEQ ID NO: 6 and VL of SEQ ID NO: 160; VH of sequence number 139 and VL of sequence number 1; VH of sequence number 139 and VL of sequence number 2; VH of sequence number 139 and VL of sequence number 3; VH of sequence number 139 and VL of sequence number 140; VH of sequence number 139 and VL of sequence number 160; VH of sequence number 159 and VL of sequence number 1; VH of sequence number 159 and VL of sequence number 2; VH of sequence number 159 and VL of sequence number 3; VH of sequence number 159 and VL of sequence number 140; VH of sequence number 159 and VL of sequence number 160; VH of sequence number 161 and VL of sequence number 1; VH of sequence number 161 and VL of sequence number 2; VH of sequence number 161 and VL of sequence number 3; VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or VH of sequence number 161 and VL of sequence number 160.

[0749] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 2 2, 23, 133, 134, 135, 136, 308, 316, 318, 319, 320 , 321, 322, 323, 324, 325, 404, 405, 406, 407, 408 , or containing a sequence of 409 amino acids.

[0750] In some embodiments, the first antigen-binding domain that binds to hK2 is the same as in Sequence ID No. 8. Contains amino acid sequence.

[0751] In some embodiments, the first antigen-binding domain that binds to hK2 is the same as in SEQ ID NO: 9 Contains amino acid sequence.

[0752] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 10. It contains the amino acid sequence.

[0753] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 11. It contains the amino acid sequence.

[0754] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 12. It contains the amino acid sequence.

[0755] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 13. It contains the amino acid sequence.

[0756] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 14. It contains the amino acid sequence.

[0757] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 15 It contains the amino acid sequence.

[0758] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 16. It contains the amino acid sequence.

[0759] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 17. It contains the amino acid sequence.

[0760] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 18 It contains the amino acid sequence.

[0761] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 19 It contains the amino acid sequence.

[0762] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 20 It contains the amino acid sequence.

[0763] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 21. It contains the amino acid sequence.

[0764] In some embodiments, the first antigen-binding domain that binds to hK2 is sequence number 22. It contains the amino acid sequence.

[0765] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 23. It contains the amino acid sequence.

[0766] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 13. It contains amino acid sequence 3.

[0767] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 13. It contains a sequence of 4 amino acids.

[0768] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 13. It contains a sequence of 5 amino acids.

[0769] In some embodiments, the first antigen-binding domain that binds to hK2 is represented by Sequence ID No. 13. It contains a sequence of 6 amino acids.

[0770] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 30 It contains an 8-amino acid sequence.

[0771] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 31 It contains a sequence of 6 amino acids.

[0772] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 31 It contains an 8-amino acid sequence.

[0773] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 31 It contains a sequence of 9 amino acids.

[0774] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 32 Contains an amino acid sequence of 0.

[0775] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 32 It contains amino acid sequence 1.

[0776] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 32 It contains the amino acid sequence 2.

[0777] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 32 It contains amino acid sequence 3.

[0778] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 32 It contains a sequence of 4 amino acids.

[0779] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 32 It contains a sequence of 5 amino acids.

[0780] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 It contains a sequence of 4 amino acids.

[0781] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 It contains a sequence of 5 amino acids.

[0782] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 It contains a sequence of 6 amino acids.

[0783] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 It contains a sequence of 7 amino acids.

[0784] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 It contains an 8-amino acid sequence.

[0785] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 It contains a sequence of 9 amino acids.

[0786] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 4 amino acid sequences and at least 80% (e.g., at least 85%, at least 90%) It contains amino acid sequences that are identical by at least 95%, or at least 99%.

[0787] In some embodiments, the first antigen-binding domain that binds to hK2 is SEQ ID NO: 40 5 amino acid sequences and at least 80% (e.g., at least 85%, at least 90%) It contains amino acid sequences that are identical by at least 95%, or at least 99%.

[0788] In certain embodiments, the first antigen-binding domain that binds to hK2 is sequence number 404 or It contains a 405-amino acid sequence. This disclosure also relates to a second antigen binding that binds to lymphocyte antigens. A domain comprising the heavy chain variable region (VH) of SEQ ID NO: 248 and the light chain variable region of SEQ ID NO: 157. Provides an antigen-binding domain that includes a variable region (VL) and binds to lymphocyte antigens. Sequence IDs 248 and 157 show the VH and VL amino acid sequences of the parent antibody CD3B815. It represents.

[0789] VL consensus sequence (sequence number 157) DIQX1TQSPX2X3LSX4SX5GX6RVX7X8X9CRARQSIG TAIHWYQQKX 10 X 11 X 12 X 13 PX 14 LLIX 15 YASESISGX 16 PSRFSGSGSGTDFTLTIX 17 SX 18 QX 19 EDX 20 AX 21 Y YCQQSX 22 SWPYTFGX 23 GTKLEIK

[0790] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is as follows: include HCDR1 (sequence number 255), HCDR2 (sequence number 256), and HCD (sequence number 257) R3, LCDR1 of sequence number 258, LCDR2 of sequence number 259, and sequence number 260 LCDR3; or HCDR1 (sequence number 255), HCDR2 (sequence number 256), and HCD (sequence number 257) R3, LCDR1 of sequence number 258, LCDR2 of sequence number 259, and sequence number 261 LCDR3.

[0791] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is as follows: include VH of sequence number 248 and VL of sequence number 249; or VH of sequence number 248 and VL of sequence number 250; or VH of sequence number 248 and VL of sequence number 251; or VH of sequence number 248 and VL of sequence number 252; or VH of sequence number 248 and VL of sequence number 253; or VH of sequence number 248 and VL of sequence number 254.

[0792] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is sequence number VH of No. 248 and at least 80% (at least 85%, at least 90%, at least VH and VL of Sequence ID No. 251 are identical by 95%, or at least 99%, and 80% (for example, at least 85%, at least 90%, at least 95%, or less) It includes VL which is 99% identical.

[0793] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is, for example, , VH of sequence number 248 and at least 80% (e.g., at least 85%, at least 9%) VH and V of Sequence ID No. 251 are 0%, at least 95%, or at least 99% identical. Includes L.

[0794] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is, for example, , VH of sequence number 248 and VL of sequence number 251 and at least 80% (for example, less V is 85%, at least 90%, at least 95%, or at least 99% identical. Includes L.

[0795] In certain embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is SEQ ID NO: 2 Includes 48 VH and 251 VL.

[0796] In some embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is as follows: include HCDR1 (sequence number 116), HCDR2 (sequence number 117), and HCD (sequence number 118) R3, LCDR1 of sequence number 119, LCDR2 of sequence number 120, and sequence number 121 LCDR3; or VH of sequence number 122 and VL of sequence number 123.

[0797] In certain embodiments, the second antigen-binding domain that binds to the lymphocyte antigen is SEQ ID NO: 2 HCDR1 at 55, HCDR2 at sequence number 256, HCDR3 at sequence number 257, sequence number LCDR1 of sequence number 258, LCDR2 of sequence number 259, and LCDR3 of sequence number 261 Includes.

[0798] In certain embodiments, the first antigen-binding domain that binds to hK2 is, respectively, SEQ ID NO: HCDR1, HCDR2, HC 170, 171, 172, 173, 174, and 175 The second component, comprising DR3, LCDR1, LCDR2, and LCDR3, binds to lymphocyte antigens. The antigen-binding domains are HCDR1 in SEQ ID NO: 255, HCDR2 in SEQ ID NO: 256, and HCDR3 at column number 257, LCDR1 at sequence number 258, LCDR2 at sequence number 259 , and LCDR3 of sequence number 261.

[0799] In certain embodiments, the first antigen-binding domain that binds to hK2 is, respectively, SEQ ID NO: HCDR1, HCDR2, HC 194, 195, 172, 173, 174, and 175 The second component, comprising DR3, LCDR1, LCDR2, and LCDR3, binds to lymphocyte antigens. The antigen-binding domains are HCDR1 in SEQ ID NO: 255, HCDR2 in SEQ ID NO: 256, and HCDR3 at column number 257, LCDR1 at sequence number 258, LCDR2 at sequence number 259 , and LCDR3 of sequence number 261.

[0800] In certain embodiments, the first antigen-binding domain that binds to hK2 is, respectively, SEQ ID NO: HCDR1, HCDR2, HC 170, 171, 172, 173, 174, and 175 The second component, comprising DR3, LCDR1, LCDR2, and LCDR3, binds to lymphocyte antigens. The antigen-binding domain includes VH in SEQ ID NO: 248 and VL in SEQ ID NO: 251.

[0801] In certain embodiments, the first antigen-binding domain that binds to hK2 is, respectively, SEQ ID NO: HCDR1, HCDR2, HC 194, 195, 172, 173, 174, and 175 The second component, comprising DR3, LCDR1, LCDR2, and LCDR3, binds to lymphocyte antigens. The antigen-binding domain includes VH in SEQ ID NO: 248 and VL in SEQ ID NO: 251.

[0802] In certain embodiments, the first antigen-binding domain that binds to hK2 is V of Sequence ID No. 162. The second antigen-binding domain, which contains H and VL of SEQ ID NO: 163, binds to lymphocyte antigens. , HCDR1 (sequence number 255), HCDR2 (sequence number 256), HCD (sequence number 257) R3, LCDR1 of sequence number 258, LCDR2 of sequence number 259, and sequence number 261 Includes LCDR3.

[0803] In certain embodiments, the first antigen-binding domain that binds to hK2 is the one in Sequence ID No. 162. It contains VH and VL of SEQ ID NO: 163, and has a second antigen-binding domain that binds to lymphocyte antigens. This includes VH of sequence number 248 and VL of sequence number 251.

[0804] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. Isolated multispecific proteins containing the main protein, which are as follows: Provide quality: The first binding domains that bind to the hK2 are sequence numbers 63, 65, 66, and 6, respectively. 7, 69, and 71 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the second domain that binds to the lymphocyte antigen is, respectively, sequence number HCDR1, HCDR2, H255, 256, 275, 258, 259, and 261 Including CDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 63, 65, 66, and 6, respectively. 7, 69, and 71 HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, the second domain which binds to the lymphocyte antigen is, respectively, sequence number HCDR1, HCDR2, H for numbers 116, 117, 118, 119, 120, and 121 Including CDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 141, 142, and 14, respectively. 3, 144, 145, and 146 HCDR1, HCDR2, HCDR3, LCDR1, It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Including CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 141, 142, and 14, respectively. 3, 144, 145, and 146 HCDR1, HCDR2, HCDR3, LCDR1, It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H, and H, respectively, with sequence numbers 116, 117, 118, 119, 120, and 121. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 188, 189, and 19, respectively. HCDR1, HCDR2, HCDR3, LCDR1, 0, 191, 192, and 193 It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; The first domains that bind to the hK2 are sequence numbers 170, 171, and 172, respectively. HCDR1, HCDR2, HCDR3, LCDR1, LC 173, 174, and 175 It includes DR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, HCD, with sequence numbers 255, 256, 257, 258, 259, and 261. Includes R2, HCDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 170, 183, and 18, respectively. 4, 185, 186, and 187 HCDR1, HCDR2, HCDR3, LCDR1, It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 176, 177, and 17, respectively. 8, 179, 180, and 181 HCDR1, HCDR2, HCDR3, LCDR1, It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 188, 189, and 19, respectively. HCDR1, HCDR2, HCDR3, LCDR1, 0, 191, 192, and 193 It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H, and H, respectively, with sequence numbers 116, 117, 118, 119, 120, and 121. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 206, 207, and 20, respectively. 8,182,470,209 HCDR1, HCDR2, HCDR3, LCDR1, LC It includes DR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, HCD with sequence numbers 116, 117, 118, 119, 120, and 121. Includes R2, HCDR3, LCDR1, LCDR2, and LCDR3; The first binding domains that bind to the hK2 are sequence numbers 206, 207, and 20, respectively. 8,182,470,209 HCDR1, HCDR2, HCDR3, LCDR1, LC It includes DR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, HCD, with sequence numbers 255, 256, 257, 258, 259, and 261. Including R2, HCDR3, LCDR1, LCDR2, and LCDR3; or The first binding domains that bind to the hK2 are sequence numbers 170, 171, and 17, respectively. 2, 173, 174, and 175 HCDR1, HCDR2, HCDR3, LCDR1, It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Includes CDR2, HCDR3, LCDR1, LCDR2, and LCDR3.

[0805] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0806] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domains, including the main, that bind to the hK2 are sequence numbers 63 and 6, respectively. 5, 66, 67, 69, and 71 HCDR1, HCDR2, HCDR3, LCDR1, It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H of sequence numbers 255, 256, 275, 258, 259, and 261, respectively. Isolation multiple specificity including CDR2, HCDR3, LCDR1, LCDR2, and LCDR3 It provides sex proteins.

[0807] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0808] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domains, including the main, that bind to the hK2 are sequence numbers 63 and 6, respectively. 5, 66, 67, 69, and 71 HCDR1, HCDR2, HCDR3, LCDR1, It comprises LCDR2 and LCDR3, and the second domain that binds to the lymphocyte antigen is HCDR1, H, and H, respectively, with sequence numbers 116, 117, 118, 119, 120, and 121. Isolation multiple specificity including CDR2, HCDR3, LCDR1, LCDR2, and LCDR3 It provides sex proteins.

[0809] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0810] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 141, HCDR1, HCDR2, HCDR3 of 142, 143, 144, 145, and 146, The second component comprises LCDR1, LCDR2, and LCDR3, which bind to the lymphocyte antigen. The main components are H with sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, This provides isolated, multispecific proteins.

[0811] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0812] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 141, HCDR1, HCDR2, HCDR3 of 142, 143, 144, 145, and 146, The second component comprises LCDR1, LCDR2, and LCDR3, which bind to the lymphocyte antigen. The main elements are H with sequence numbers 116, 117, 118, 119, 120, and 121, respectively. Including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, This provides isolated, multispecific proteins.

[0813] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0814] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 188, HCDR1, HCDR2, HCDR3 of 189, 190, 191, 192, and 193, The second component comprises LCDR1, LCDR2, and LCDR3, which bind to the lymphocyte antigen. The main components are H with sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, This provides isolated, multispecific proteins.

[0815] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0816] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first domains that include the main and bind to the hK2 are sequence numbers 170 and 17, respectively. HCDR1, HCDR2, HCDR3, LC 1, 172, 173, 174, and 175 A second domain comprising DR1, LCDR2, and LCDR3 that binds to the lymphocyte antigen These are HCDs with sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Isolated, containing R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. It provides multispecific proteins.

[0817] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0818] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 170, HCDR1, HCDR2, HCDR3 of 183, 184, 185, 186, and 187, The second component comprises LCDR1, LCDR2, and LCDR3, which bind to the lymphocyte antigen. The main components are H with sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, This provides isolated, multispecific proteins.

[0819] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0820] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 176, HCDR1, HCDR2, HCDR3 of 177, 178, 179, 180, and 181, The second component comprises LCDR1, LCDR2, and LCDR3, which bind to the lymphocyte antigen. The main components are H with sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, This provides isolated, multispecific proteins.

[0821] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0822] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 188, HCDR1, HCDR2, HCDR3 of 189, 190, 191, 192, and 193, The second component comprises LCDR1, LCDR2, and LCDR3, which bind to the lymphocyte antigen. The main elements are H with sequence numbers 116, 117, 118, 119, 120, and 121, respectively. Including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, This provides isolated, multispecific proteins.

[0823] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0824] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 206, HCDR1, HCDR2, HCDR3, LC 207, 208, 182, 470, 209 A second domain comprising DR1, LCDR2, and LCDR3 that binds to the lymphocyte antigen These are HCDs with sequence numbers 116, 117, 118, 119, 120, and 121, respectively. Isolated, containing R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. It provides multispecific proteins.

[0825] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0826] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 206, HCDR1, HCDR2, HCDR3, LC 207, 208, 182, 470, 209 A second domain comprising DR1, LCDR2, and LCDR3 that binds to the lymphocyte antigen These are HCDs with sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Isolated, containing R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. It provides multispecific proteins.

[0827] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0828] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, including the main, that binds to the hK2 is sequence number 170, HCDR1, HCDR2, HCDR3 of 171, 172, 173, 174, and 175, The second component comprises LCDR1, LCDR2, and LCDR3, which bind to the lymphocyte antigen. The main components are H with sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Including CDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, This provides isolated, multispecific proteins.

[0829] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0830] This disclosure also relates to a first domain that binds to hK2 and a second anti- An isolated multispecific protein containing a primordial domain, wherein the isolated multispecific protein is as follows: Provides protein: The first binding domain that binds to the hK2 is VH of SEQ ID NO: 139 and SEQ ID NO: 14 It contains VL of 0, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of sequence number 251; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 139 and SEQ ID NO: 14 It contains VL of 0, and the second binding domain that binds to the lymphocyte antigen is of SEQ ID NO: 122 Includes VH and VL of sequence number 123; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 137 and SEQ ID NO: 13 It contains 8 VLs, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of sequence number 251; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 137 and SEQ ID NO: 13 It contains 8 VLs, and the second binding domain that binds to the lymphocyte antigen is the same as in SEQ ID NO: 122 Includes VH and VL of sequence number 123; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 168 and SEQ ID NO: 16 It contains 9 VLs, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of sequence number 251; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 162 and SEQ ID NO: 16 It contains VL 3, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of sequence number 251; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 166 and SEQ ID NO: 44 It contains 4 VL, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of sequence number 251; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 164 and SEQ ID NO: 16 It contains VL of 5, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of sequence number 251; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 168 and SEQ ID NO: 16 It contains 9 VLs, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 122 Includes VH and VL of sequence number 123; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 204 and SEQ ID NO: 20 It contains VL of 5, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 122 Includes VH and VL of sequence number 123; The first binding domain that binds to the hK2 is VH of SEQ ID NO: 204 and SEQ ID NO: 20 It contains VL of 5, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of Sequence ID No. 251; or The first binding domain that binds to the hK2 is VH of SEQ ID NO: 162 and SEQ ID NO: 16 It contains VL 3, and the second binding domain that binds to the lymphocyte antigen is SEQ ID NO: 248 Includes VH and VL of sequence number 251.

[0831] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0832] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH of SEQ ID NO: 139 and The sequence contains the VL of sequence number 140, and the second binding domain that binds to the lymphocyte antigen is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0833] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0834] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH of SEQ ID NO: 139 and The sequence contains the VL of sequence number 140, and the second binding domain that binds to the lymphocyte antigen is sequence This provides isolated multispecific proteins, including VH (number 122) and VL (sequence number 123). ru.

[0835] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0836] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The sequence contains the VL of sequence number 138, and the second binding domain that binds to the lymphocyte antigen is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0837] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0838] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The sequence contains the VL of sequence number 138, and the second binding domain that binds to the lymphocyte antigen is sequence This provides isolated multispecific proteins, including VH (number 122) and VL (sequence number 123). ru.

[0839] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0840] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The sequence contains the VL of sequence number 169, and the second binding domain that binds to the lymphocyte antigen is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0841] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0842] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The second binding domain, which contains the VL of sequence number 163 and binds to the lymphocyte antigen, is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0843] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0844] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The second binding domain, which contains the VL of sequence number 444 and binds to the lymphocyte antigen, is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0845] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0846] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH of SEQ ID NO: 164 and The second binding domain, which contains the VL of sequence number 165 and binds to the lymphocyte antigen, is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0847] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0848] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The sequence contains the VL of sequence number 169, and the second binding domain that binds to the lymphocyte antigen is sequence This provides isolated multispecific proteins, including VH (number 122) and VL (sequence number 123). ru.

[0849] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0850] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The second binding domain, which contains the VL of sequence number 205 and binds to the lymphocyte antigen, is sequence This provides isolated multispecific proteins, including VH (number 122) and VL (sequence number 123). ru.

[0851] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0852] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The second binding domain, which contains the VL of sequence number 205 and binds to the lymphocyte antigen, is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0853] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It's about quality.

[0854] This disclosure also relates to a first domain that binds to hK2 and a second domain that binds to lymphocyte antigens. The first binding domain, which includes the main and binds to the hK2, is VH and The second binding domain, which contains the VL of sequence number 163 and binds to the lymphocyte antigen, is sequence This provides isolated multispecific proteins, including VH (number 248) and VL (sequence number 251). ru.

[0855] In some embodiments, isolated multispecific proteins are anti-hK2 / anti-CD3 proteins. It is of quality. In some embodiments, the first antigen-binding domain that binds to hK2 is the first Conjugate to the immunoglobulin (Ig) constant region or a fragment of the first Ig constant region. The second antigen-binding domain, which binds to and / or lymphocyte antigens, is the second immunoglobulin. It is conjugated into a brin (Ig) steady-state region or a fragment of a second Ig steady-state region.

[0856] In some embodiments, a fragment of the first Ig steady-state region and / or the second Ig steady-state region The fragment includes the Fc region.

[0857] In some embodiments, a fragment of the first Ig steady-state region and / or the second Ig steady-state region The fragment contains the CH2 domain.

[0858] In some embodiments, a fragment of the first Ig steady-state region and / or the second Ig steady-state region The fragment contains the CH3 domain.

[0859] In some embodiments, a fragment of the first Ig steady-state region and / or the second Ig steady-state region The fragment contains the CH2 domain and the CH3 domain.

[0860] In some embodiments, a fragment of the first Ig steady-state region and / or the second Ig steady-state region The fragment includes at least a portion of the hinge, the CH2 domain, and the CH3 domain.

[0861] In some embodiments, the Ig steady-state region fragment comprises a hinge, a CH2 domain, and a CH2 domain. Includes 3 domains.

[0862] In some embodiments, the multispecific protein is the first antigen-binding protein that binds to hK2. Between the domain and the first Ig constant region or a fragment of the first Ig constant region, and lymphocyte antigens A second antigen-binding domain that binds to and a second Ig constant region or a fragment of the second Ig constant region. A second linker (L2) is further included between them.

[0863] In some embodiments, L2 is represented by sequence numbers 7, 76, 77, 78, 79, 80, 81. , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 1 It contains the amino acid sequence 06, 107, or 108.

[0864] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region, and The Ig constant region of 2 or the fragment of the second Ig constant region consists of IgG1, IgG2, and IgG3 , or IgG4 isotype.

[0865] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG1 isotype.

[0866] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG2 isotype.

[0867] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG3 isotype.

[0868] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region is an IgG4 isotype.

[0869] In certain embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second I The IgG steady-state region or the fragment of the second IgG steady-state region is an IgG1 isotype.

[0870] A first Ig steady-state region or a fragment of the first Ig steady-state region and a second Ig steady-state region or the second The Ig constant region fragment may be further genetically engineered as described herein.

[0871] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region, and The Ig constant region of the second Ig constant region or a fragment of the second Ig constant region is transferred to the FcγR of the multispecific protein. It includes at least one mutation that reduces binding.

[0872] In some embodiments, the binding of multispecific proteins to FcγR is reduced. Each mutation is F234A / L235A, L234A / L235A, L234A / L 235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L23 5A, N297A, V234A / G237A, K214T / E233P / L234V / L 235A / G236 Missing / A327G / P331A / D365E / L358M, H268 Q / V309L / A330S / P331S, S267E / L328F, L234F / L2 35E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238 S, and S228P / F234A / L235A / G236 (missing) / G237A / P238S Selected from the group consisting of, the numbering of residues follows the EU index. In certain embodiments, The first Ig steady-state region or a fragment of the first Ig steady-state region and / or the second Ig steady-state region Alternatively, the second Ig constant region fragment has the following mutation: L234A_L235A_D265S Includes.

[0873] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region, and The Ig constant region of 2 or a fragment of the second Ig constant region is the Fcγ receptor of the multispecific protein. It contains at least one mutation that enhances binding to the body (FcγR).

[0874] In some embodiments, the binding of multispecific proteins to FcγR is enhanced. Each of the mutations is S239D / I332E, S298A / E333A / K334A, F 243L / R292P / Y300L, F243L / R292P / Y300L / P396L F243L / R292P / Y300L / V305I / P396L, and G236A / S The residues are selected from the group consisting of 239D / I332E, and the numbering follows the EU index.

[0875] In some embodiments, FcγR is FcγRI, FcγRIIA, FcγRIIB , or FcγRIII, or any combination thereof.

[0876] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region, and The Ig constant region of the second Ig constant region or a fragment of the second Ig constant region adjusts the half-life of the multispecific protein. It contains at least one mutation that makes up a node.

[0877] In some embodiments, at least one of the multispecific proteins regulates the half-life of the multispecific protein. The mutations are H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H433K / N434F, T308P / N434A, and H4 Selected from a group consisting of 35R residues, the residue numbering follows the EU index.

[0878] In some embodiments, the multispecific protein has a CH3 domain in the first Ig constant region. At least one mutation in the CH3 domain of the Ig or first Ig constant region fragment. and / or the CH3 domain of the second Ig steady-state region or the C of a fragment of the second Ig steady-state region Contains at least one mutation in the H3 domain.

[0879] In some embodiments, the CH3 domain of the first Ig steady region or the first Ig constant At least one mutation in the CH3 domain of the constitutive region fragment and / or a second Ig stationary region A small amount of CH3 domain in the region or in the CH3 domain of the fragment of the second Ig constant region At least one mutation is T350V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T394W, T394S, Y407T, Y407A, T366 S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K3 92M / T394W, F405A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T366A / K409F, L351Y / Y407A, T366 V / K409F, T366A / K409F, T350V / L351Y / F405A / Y4 Selected from the group consisting of 07V and T350V / T366L / K392L / T394W The numbering of residues follows the EU index. In certain embodiments, the first Ig constant region or The first Ig constant region fragment contains the following mutation: T350V_T366L_K39 2L_T394W and / or the second Ig steady-state region or the fragment of the second Ig steady-state region are This includes the following mutations: T350V_L351Y_F405A_Y407V.

[0880] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region contains the following mutations: L234A_L235A_D265S_T350V_L3 in the first steady-state Ig region 51Y_F405A_Y407V and L235A_L235 in the second Ig steady-state region A_D265S_T350V_T366L_K392L_T394W; or L235A_L235A_D265S_T350V_T3 in the first steady-state Ig region 66L_K392L_T394W and L235A_L235 in the second Ig steady-state region A_D265S_T350V_L351Y_F405A_Y407V.

[0881] In some embodiments, a first Ig steady-state region or a fragment of the first Ig steady-state region and a second The Ig constant region or a fragment of the second Ig constant region contains the following mutations: L234A_L235A_D265S_T350V_L3 in the first steady-state Ig region 51Y_F405A_Y407V and L234A_L235 in the second Ig steady-state region A_D265S_T350V_T366L_K392L_T394W; or L234A_L235A_D265S_T350V_T3 in the first steady-state Ig region 66L_K392L_T394W and L234A_L235 in the second Ig steady-state region A_D265S_T350V_L351Y_F405A_Y407V.

[0882] In some embodiments, the first Ig heavy chain steady region or a fragment of the first Ig heavy chain steady region This is sequence number 378 and at least 80% (for example, at least 85%, at least 90%) , containing amino acid sequences that are identical by at least 95%, or at least 99%.

[0883] In certain embodiments, the first Ig heavy chain steady region or a fragment of the first Ig heavy chain steady region is Contains an amino acid sequence identical to sequence number 378.

[0884] In some embodiments, the first Ig light chain steady region or a fragment of the first Ig light chain steady region This is at least 80% (e.g., at least 85%, at least 90%) of sequence number 309. , containing amino acid sequences that are identical by at least 95%, or at least 99%.

[0885] In a particular embodiment, the first Ig light chain steady region or a fragment of the first Ig light chain steady region is Contains an amino acid sequence identical to sequence number 309.

[0886] In some embodiments, the second Ig steady region or fragment of the second Ig steady region is an array Number 109 and at least 80% (for example, at least 85%, at least 90%, less) They both contain amino acid sequences that are 95% or at least 99% identical.

[0887] In certain embodiments, the second Ig steady-state region or a fragment of the second Ig steady-state region is, It contains an amino acid sequence identical to that of 109.

[0888] In certain embodiments, the first Ig heavy chain constant region is the same as that of SEQ ID NO: 378. The amino acid sequence includes an acid sequence, and the first Ig light chain constant region has the same amino acid sequence as SEQ ID NO: 309. The second Ig constant region contains an amino acid sequence identical to that of SEQ ID NO: 109.

[0889] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain and the first Ig weight containing the same amino acid sequence as SEQ ID NO: 378 It includes a chain-steady region or a fragment of the first Ig heavy chain-steady region.

[0890] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain and the first Ig cell containing the same amino acid sequence as SEQ ID NO: 309 It includes a chain steady region or a fragment of the first Ig light chain steady region.

[0891] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. (i) The antigen-binding domain and the first I containing the same amino acid sequence as SEQ ID NO: 378 (ii) a fragment of the g heavy chain steady region or the first Ig heavy chain steady region, and the same as SEQ ID NO: 309 A first Ig light chain constant region or a fragment of the first Ig light chain constant region containing a certain amino acid sequence include.

[0892] In certain embodiments, the isolated proteins disclosed herein are lymphocyte antigens (e.g., A second antigen-binding domain that binds to CD3, and an amino acid identical to that of Sequence ID No. 109. It contains a second Ig constant region containing an acid sequence or a fragment of the second Ig constant region.

[0893] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. (i) The antigen-binding domain and the first I containing the same amino acid sequence as SEQ ID NO: 378 (ii) a fragment of the g heavy chain steady region or the first Ig heavy chain steady region, and the same as SEQ ID NO: 309 A first Ig light chain constant region or a fragment of the first Ig light chain constant region containing a certain amino acid sequence The isolated protein contains a second antigenic binding to lymphocyte antigens (e.g., CD3). A synthetic domain and a second Ig constant region containing the same amino acid sequence as SEQ ID NO: 109 or It further includes a fragment of the second Ig steady-state region.

[0894] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and The first antigen-binding domain that binds to hK2, including LCDR3, is identical to SEQ ID NO: 378. A first Ig heavy chain constant region or a fragment of the first Ig heavy chain constant region containing the amino acid sequence Includes.

[0895] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and The first antigen-binding domain that binds to hK2, including LCDR3, is identical to SEQ ID NO: 309. A first Ig light chain constant region or a fragment of the first Ig light chain constant region containing the amino acid sequence and Includes.

[0896] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and (i)SEQ ID NO: 378 A first Ig heavy chain constant region containing an amino acid sequence identical to that of the first Ig heavy chain constant region (ii) the fragment and the first Ig light chain constant region containing the same amino acid sequence as SEQ ID NO: 309 It includes a region or a fragment of the first Ig light chain constant region.

[0897] In certain embodiments, the isolated proteins disclosed herein are lymphocyte antigens (e.g., A second antigen-binding domain that binds to CD3, and is the HCDR1 of sequence number 255. HCDR2 (sequence number 256), HCDR3 (sequence number 257), LCDR (sequence number 258) 1. Lymphocyte anti- It contains a second antigen-binding domain that binds to the source, and an amino acid sequence identical to that of sequence number 109. It includes a second Ig steady-state region or a fragment of the second Ig steady-state region.

[0898] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domains are, respectively, sequence numbers 170, 171, 172, 173, and 17 4, and 175 HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and (i)SEQ ID NO: 378 A first Ig heavy chain constant region containing an amino acid sequence identical to that of the first Ig heavy chain constant region (ii) the fragment and the first Ig light chain constant region containing the same amino acid sequence as SEQ ID NO: 309 The isolated protein comprises a region or a fragment of the first Ig light chain constant region, and the isolated protein is a lymphocyte antigen ( For example, a second antigen-binding domain that binds to CD3, and the HCD of sequence number 255. R1, HCDR2 (sequence number 256), HCDR3 (sequence number 257), L (sequence number 258) Including CDR1, LCDR2 of sequence number 259, and LCDR3 of sequence number 261, A second antigen-binding domain that binds to the Pacyl antigen, and an amino acid sequence identical to that of SEQ ID NO: 109. Further comprising a second Ig steady-state region or a fragment of the second Ig steady-state region containing a column.

[0899] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and the amino acid sequence identical to that of SEQ ID NO: 378. It includes a first Ig heavy chain steady region or a fragment of the first Ig heavy chain steady region.

[0900] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and the amino acid sequence identical to that of SEQ ID NO: 309. It comprises a first Ig light chain constant region or a fragment of the first Ig light chain constant region.

[0901] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and (i) an amino acid identical to that of Sequence ID No. 378. (ii) a first Ig heavy chain constant region containing an acid sequence or a fragment of the first Ig heavy chain constant region, and The first Ig light chain constant region or the first Ig light chain containing the same amino acid sequence as column number 309 It includes fragments of the chain-steady region.

[0902] In certain embodiments, the isolated proteins disclosed herein are lymphocyte antigens (e.g., A second antigen-binding domain that binds to CD3, and the amino acid sequence of SEQ ID NO: 331 It includes a second antigen-binding domain that binds to lymphocyte antigens, and is identical to SEQ ID NO: 109. It contains a second Ig constant region or a fragment of a second Ig constant region that includes a certain amino acid sequence.

[0903] In certain embodiments, the isolated protein disclosed herein is a first protein that binds to hK2. The antigen-binding domain includes VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163, The first antigen-binding domain that binds to hK2, and (i) an amino acid identical to that of Sequence ID No. 378. (ii) a first Ig heavy chain constant region containing an acid sequence or a fragment of the first Ig heavy chain constant region, and The first Ig light chain constant region or the first Ig light chain containing the same amino acid sequence as column number 309 The isolated protein comprises a fragment of the chain constant region, and the isolated protein is a lymphocyte antigen (e.g., CD3). A second antigen-binding domain that binds, comprising the amino acid sequence of SEQ ID NO: 331, A second antigen-binding domain that binds to the Pacyl antigen, and an amino acid sequence identical to that of SEQ ID NO: 109. Further comprising a second Ig steady-state region or a fragment of the second Ig steady-state region containing a column.

[0904] Production of multispecific proteins containing antigen-binding fragments that bind to hK2 The antigen-binding fragment that binds to hK2 in this disclosure may be genetically modified into a multispecific antibody. Such antibodies are also included within the scope of the present invention.

[0905] Antigen-binding fragments that bind to hK2 were prepared using Fab arm exchange, in vitro Two monospecific divalent antibodies within the Ig constant region CH3 domain promote Fab arm replacement. Genetic engineering may be performed on full-length, multispecific antibodies in which substitutions have been introduced into the body. , having a specific substitution in the CH3 domain that promotes the stability of the heterodimer, Monospecific bivalent antibodies are genetically engineered. These antibodies have cysteine ​​in the hinge region. Under sufficient reducing conditions to isomerize the disulfide bond, both are incubated. This process is performed, and as a result, bispecific antibodies are generated by exchanging the Fab arm. The Bate condition can, optimally, be returned to a non-reducing condition. Two typical reducing agents that can be used are: - Mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioethylamine Slitol (DTE), glutathione, tris(2-carboxyethyl)phosphine (T The CEP is L-cysteine, and β-mercaptoethanol, preferably 2-mercaptoethanol. Lucaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphorus A reducing agent selected from the group consisting of fins. For example, at a temperature of at least 20°C, At least 25 mM 2-MEA or at least 0.5 mM dithiothreitol In the presence of [unclear], at pH 5-8, for example, pH 7.0 or pH 7.4, for at least 90 minutes An incubator can be used.

[0906] Possible CH3 mutations to use include the knob-in-hole mutation (Genentech) and electrostatic mutation. Targeted matching mutations (Chugai, Amgen, NovoNordisk, Oncomed) , Strand exchange gene manipulation domain body (SEEDbody) (EMD Serono), D uobody(registered trademark) mutation (Genmab), and other asymmetric mutations (e.g., Zym Technologies such as eworks can be cited.

[0907] The nobu-in-hole mutation is disclosed, for example, in International Pu...

Claims

1. An isolated cyanotype containing an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2) Protein, wherein the antigen-binding domain is sequence numbers 111, 112, 113, 114 , and bind to an epitope on hK2 having a sequence selected from the group consisting of 115, Isolated protein.

2. The antigen-binding domains that bind to hK2 are KVTEF (SEQ ID NO: 111) and HYR The following is a claim that binds to hK2 in an epitope having the sequence KW (sequence number 112): Isolated protein.

3. The antigen-binding domain that binds to hK2 competes with the reference antibody for binding to hK2. The isolated protein according to claim 1, wherein the reference antibody comprises the following: a. HCDR1, HCDR2, and HCDR3 of VH in sequence number 137, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 138; or b. HCDR1, HCDR2, and HCDR3 of VH in sequence number 162, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 163; or c. HCDR1, HCDR2, and HCDR3 of VH in sequence number 164, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 165; or d. HCDR1, HCDR2, and HCDR3 of VH of sequence number 166, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 167; or e. HCDR1, HCDR2, and HCDR3 of VH in sequence number 168, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 169; or f. HCDR1, HCDR2, and HCDR3 of VH in sequence number 204, and sequence number LCDR1, LCDR2, and LCDR3 of VL No. 205; or g. HCDR1, HCDR2, and HCDR3 of VH in sequence number 166, and sequence number LCDR1, LCDR2, and LCDR3 of VL No.

444.

4. The antigen-binding domain that binds to hK2 competes with the reference antibody for binding to hK2. The reference antibody is HCDR1, HCDR2, and HCDR3 of the VH of SEQ ID NO:

162. The claim further includes LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No.

163. The isolated protein described in 3.

5. The following HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR The isolated protein according to claims 1 to 4, comprising 3. a. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; b. Sequence IDs 170, 171, 172, 173, 174, and 175, respectively; c. Sequence IDs 176, 177, 178, 179, 180, and 181, respectively; d. Sequence IDs 170, 183, 184, 185, 186, and 187, respectively; e. Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; f. Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; g. Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; h. Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; i. Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; j. Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; k. Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or l. Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

6. HCDR1 of sequence numbers 170, 171, 172, 173, 174, and 175, respectively. Claim 5, which includes HCDR1, HCDR3, LCDR1, LCDR2, and LCDR3. The isolated protein described above.

7. The antigen-binding domain that binds to the aforementioned hK2 is scFv, (scFv) ２ , Fv, Fab , F(ab') ２ Fd, dAb, or VHH as described in any one of claims 1 to 6. Isolated protein.

8. The isolated tangent according to claim 7, wherein the antigen-binding domain that binds to hK2 is Fab. Pak quality.

9. The isolated tangent to the aforementioned hK2 is VHH, according to claim 7. Pak quality.

10. The isolation agent according to claim 7, wherein the antigen-binding domain that binds to hK2 is scFV. Protein.

11. The scFv is VH, the first linker (L1), and from the N-terminus to the C-terminus, VL (VH-L1-VL), or including VL, L1, and VH (VL-L1-VH), The isolated protein described in item 10.

12. The isolated protein according to claim 11, wherein L1 comprises the following a. Approximately 5 to 50 amino acids, b. Approximately 5 to 40 amino acids, c. Approximately 10 to 30 amino acids, or d. Approximately 10 to 20 amino acids.

13. The aforementioned L1 corresponds to sequence numbers 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, ８５、８６、８７、８８、８９、９０、９１、９２、９３、９４、９５、９６、９７、９ 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 1 The isolated protein according to claim 12, comprising the amino acid sequence 08.

14. The isolated protein according to claim 13, wherein L1 comprises the amino acid sequence of SEQ ID NO:

7.

15. The antigen-binding domain that binds to the aforementioned hK2 is sequence numbers 137, 162, 164, and 166. , 168 or 204 VH and sequence numbers 138, 163, 165, 167, 169, or The isolated protein according to any one of claims 1 to 14, comprising 205 VL.

16. The isolated antigen according to claim 15, comprising the following antigen-binding domain that binds to hK2. Protein: a. VH of SEQ ID NO: 137 and VL of SEQ ID NO: 138; or b. VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; or c. VH of sequence number 164 and VL of sequence number 165; or d. VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167; or e. VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169; or f. VH of sequence number 204 and VL of sequence number 205; or g. VH of sequence number 166 and VL of sequence number 444.

17. The antigen-binding domain that binds to hK2 is at least 80% of the VH of Sequence ID No.

162. (For example, at least 85%, at least 90%, at least 95%, at least 99%) VH and VL of Sequence ID No. 163 are identical (or 100%) and at least 80% (for example) , at least 85%, at least 90%, at least 95%, at least 99%, or 1 The isolated protein according to claim 15, comprising VL which is identical to 00%).

18. The antigen-binding domain that binds to hK2 is sequence numbers 133, 134, 308, and 316. A group consisting of 324, 325, 404, 405, 406, 407, 408, and 409. The isolated protein according to any one of claims 1 to 17, comprising an amino acid sequence selected from quality.

19. The antigen-binding domain that binds to hK2 is the amino acid sequence of SEQ ID NO: 404 or 405. and at least 80% (for example, at least 85%, at least 90%, at least 95%) Claims 1 to 17 include amino acid sequences that are identical by at least 99%, or 100%, respectively. The isolated protein described in any one of the items.

20. An isolated cyanotype containing an antigen-binding domain that binds to kallikrein-related peptidase 2 (hK2) The protein is such that the antigen-binding domain that binds to hK2 is the heavy chain variable of SEQ ID NO: 75 An isolated protein containing the VH region and the VL light chain variable region of SEQ ID NO:

74.

21. The antigen-binding domain that binds to hK2 is sequence numbers 4, 5, 6, 139, 159, or This includes VH of 161 and VL of sequence numbers 1, 2, 3, 140, or 160, provided that the previous The antigen-binding domain that binds to hK2 is both VH of SEQ ID NO: 5 and VL of SEQ ID NO: 2 The isolated protein according to claim 20, which does not contain

22. The isolated antigen according to claim 21, comprising the following antigen-binding domain that binds to hK2. Protein: a. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1; b. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2; c. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3; d. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; e. VH of sequence number 4 and VL of sequence number 160; f. VH of sequence number 5 and VL of sequence number 1; g. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; h. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; i. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; j. VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1; k. VH of sequence number 6 and VL of sequence number 2; l. VH of sequence number 6 and VL of sequence number 3; m. VH of sequence number 6 and VL of sequence number 140; n. VH of sequence number 6 and VL of sequence number 160; o. VH of sequence number 139 and VL of sequence number 1; p. VH of sequence number 139 and VL of sequence number 2; q. VH of sequence number 139 and VL of sequence number 3; r. VH of sequence number 139 and VL of sequence number 140; s. VH of sequence number 139 and VL of sequence number 160; t. VH of sequence number 159 and VL of sequence number 1; u. VH of sequence number 159 and VL of sequence number 2; v. VH of sequence number 159 and VL of sequence number 3; w. VH of sequence number 159 and VL of sequence number 140; x. VH of sequence number 159 and VL of sequence number 160; y. VH of sequence number 161 and VL of sequence number 1; z. VH of sequence number 161 and VL of sequence number 2; aa. VH of sequence number 161 and VL of sequence number 3; bb. VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or cc. VH of sequence number 161 and VL of sequence number 160.

23. The following HCDR1, HCDR1, HCDR3, LCDR1, LCDR2, and LCDR The isolated protein according to any one of claims 20 to 22, comprising 3. a. Sequence IDs 63, 64, 66, 67, 69, and 71, respectively; b. Sequence IDs 63, 65, 66, 68, 70, and 71, respectively; c. Sequence IDs 63, 65, 66, 67, 69, and 71, respectively; d. Sequence IDs 72, 73, 66, 67, 69, and 71, respectively; or e. Sequence IDs 72, 73, 66, 68, 70, and 71, respectively; or f. Sequence numbers 72, 73, 66, 67, 69, and 71, respectively.

24. The antigen-binding domain that binds to the aforementioned hK2 is scFv, (scFv) ２ , Fv, Fab , F(ab') ２ The isolated protein according to claim 21, which is Fd, dAb, or VHH quality.

25. The isolation agent according to claim 24, wherein the antigen-binding domain that binds to hK2 is Fab. Protein.

26. The isolation agent according to claim 24, wherein the antigen-binding domain that binds to hK2 is VHH. Protein.

27. The isolation according to claim 24, wherein the antigen-binding domain that binds to hK2 is scFV. protein.

28. The scFv is VH, the first linker (L1), and from the N-terminus to the C-terminus, VL (VH-L1-VL), or including VL, L1, and VH (VL-L1-VH), The isolated protein described in item 27.

29. The isolated protein according to claim 28, wherein L1 comprises the following a. Approximately 5 to 50 amino acids, b. Approximately 5 to 40 amino acids, c. Approximately 10 to 30 amino acids, d. Or about 10 to 20 amino acids.

30. The aforementioned L1 corresponds to sequence numbers 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, ８５、８６、８７、８８、８９、９０、９１、９２、９３、９４、９５、９６、９７、９ 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 1 The isolated protein according to claim 29, comprising the amino acid sequence 08.

31. The antigen-binding domain that binds to hK2 is sequence numbers 8, 9, 10, 11, 12, and 13. 、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、１３５、１３６、３ Claim 21, comprising the amino acid sequence 18, 319, 320, 321, 322, or 323 Isolated protein as described in any one of items ~30.

32. The isolated protein according to any one of claims 1 to 31, which is a single-specific protein. 。

33. The isolated protein according to any one of claims 1 to 31, which is a multispecific protein. 。

34. The isolation method according to claim 33, wherein the multispecific protein is a bispecific protein. protein.

35. The isolation according to claim 33, wherein the multispecific protein is a triplespecific protein. protein.

36. The multiplicity according to claim 34 or 35, comprising an antigen-binding domain that binds to an antigen on a lymphocyte. Heavy specific protein.

37. The multispecific protein according to claim 36, wherein the lymphocyte is a T cell.

38. The T cell is CD8 ＋ The multispecific protein according to claim 37, which is a T cell.

39. The multiple specificity according to claim 38, wherein the lymphocytes are natural killer (NK) cells. Sex protein.

40. The antigens on the lymphocytes are CD3, CD3 epsilon (CD3ε), CD8, KI2L 4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD -1, CD195, or NKG2C, the multiple specificity according to any one of claims 36 Sex protein.

41. The multispecific protein according to claim 40, wherein the antigen on the lymphocyte is CD3ε. 。

42. The multiplicity feature according to claim 41, wherein the antigen-binding domain that binds to the CD3ε includes the following: Isomer proteins: a. Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 116, HCD of SEQ ID NO: 117 R2, HCDR3 of SEQ ID NO: 118, Light chain complementarity determination region 1 (LCDR of SEQ ID NO: 119) 1) LCDR2 of SEQ ID NO: 120, and LCDR3 of SEQ ID NO: 121; or b. VH of sequence number 122 and VL of sequence number 123.

43. The antigen-binding domains that bind to the aforementioned CD3ε are VH of SEQ ID NO: 248 and SEQ ID NO: 15 The multispecific protein according to claim 41, comprising VL 7.

44. The multiplicity feature according to claim 43, wherein the antigen-binding domain that binds to the CD3ε includes the following: Isogamous proteins: a. Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 255, HCD of SEQ ID NO: 256 R2, HCDR3 of SEQ ID NO: 257, light chain complementarity determination region 1 (LCDR of SEQ ID NO: 258) 1) LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 260; or b. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, H of SEQ ID NO: 257 CDR3, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and SEQ ID NO: 2 61 LCDR3; or c. VH of sequence number 248 and VL of sequence number 249; or d. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 250; or e. VH of sequence number 248 and VL of sequence number 251; or f. VH of sequence number 248 and VL of sequence number 252; or g. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 253; or h. VH of sequence number 248 and VL of sequence number 254.

45. The antigen-binding domain that binds to the aforementioned CD3ε is HCDR1 of SEQ ID NO. 255, SEQ ID NO. HCDR2 at sequence number 256, HCDR3 at sequence number 257, LCDR1 at sequence number 258, sequence The claim 43 includes LCDR2 number 259 and LCDR3 number 261. Multiple specificity proteins.

46. The antigen-binding domain that binds to the CD3ε is at least 80% of the VH of Sequence ID No.

248. % (for example, at least 85%, at least 90%, at least 95%, at least 99%) VH and VL of Sequence ID No. 251 are identical in % or 100% of the original (e.g., at least 80%) If so, at least 85%, at least 90%, at least 95%, at least 99%, or The multispecific protein according to claim 43 or 45, comprising VL which is 100% identical.

47. The binding domains that bind to hK2 are the following HCDR1, HCDR2, HCDR3, The following is a description of any one of claims 41 to 46, including LCDR1, LCDR2, and LCDR3. The multispecific proteins a. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; b. Sequence IDs 170, 171, 172, 173, 174, and 175, respectively; c. Sequence IDs 176, 177, 178, 179, 180, and 181, respectively; d. Sequence IDs 170, 183, 184, 185, 186, and 187, respectively; e. Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; f. Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; g. Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; h. Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; i. Sequence numbers 196, 197, 178, 179, 190, and 181, respectively; j. Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; k. Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; l. Sequence IDs 216, 217, 218, 182, 470, and 209, respectively; m. Sequence numbers 63, 64, 66, 67, 69, and 71, respectively; n. Sequence IDs 63, 65, 66, 68, 70, and 71, respectively; o. Sequence numbers 72, 73, 66, 67, 69, and 71, respectively; p. Sequence IDs 72, 73, 66, 68, 70, and 71, respectively; q. VH of sequence number 137 and VL of sequence number 138; r. VH of sequence number 162 and VL of sequence number 163; s. VH of sequence number 164 and VL of sequence number 165; t. VH of sequence number 166 and VL of sequence number 167; u. VH of sequence number 168 and VL of sequence number 169; v. VH of sequence number 204 and VL of sequence number 205; w. VH of sequence number 166 and VL of sequence number 444; x. VH of sequence number 4 and VL of sequence number 3; y. VH of sequence number 139 and VL of sequence number 140; z. VH of sequence number 159 and VL of sequence number 140; aa. VH of sequence number 159 and VL of sequence number 160; or bb. VH of sequence number 161 and VL of sequence number 160.

48. The binding domains that bind to hK2 are, respectively, sequence numbers 170, 171, 172, and 1. 73, 174, and 175 HCDR1, HCDR2, HCDR3, LCDR1, LCD The multispecific protein according to claim 47, comprising R2 and LCDR3.

49. Any one of claims 41 to 48, wherein the binding domain that binds to the CD3 includes the following: The multispecific proteins described a. HCDR1 of SEQ ID NO: 116, HCDR2 of SEQ ID NO: 117, H of SEQ ID NO: 118 CDR3, LCDR1 of SEQ ID NO: 119, LCDR2 of SEQ ID NO: 120, and SEQ ID NO: 1 21 LCDR3; or b. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, H of SEQ ID NO: 257 CDR3, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and SEQ ID NO: 2 60 LCDR3; or c. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, H of SEQ ID NO: 257 CDR3, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and SEQ ID NO: 2 61 LCDR3; or d. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 249; or e. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 250; or f. VH of sequence number 248 and VL of sequence number 251; or g. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 252; or h. VH of sequence number 248 and VL of sequence number 253; or i. VH of sequence number 248 and VL of sequence number 254; or j. VH of sequence number 122 and VL of sequence number 123.

50. The single unit according to any one of claims 1 to 49, which is conjugated to the half-life extension portion. Protein release.

51. The half-life extension portion comprises immunoglobulin (Ig), a fragment of Ig, the Ig constant region, and The Ig constant region fragment, Fc region, transferrin, albumin, albumin-binding dormant The isolated protein according to claim 50, wherein it is 100% or polyethylene glycol.

52. The isolated protein according to claim 51, wherein the fragment of the Ig constant region includes an Fc region.

53. The isolated protein according to claim 52, wherein the fragment of the Ig constant region includes a CH2 domain. quality.

54. The isolated protein according to claim 52, wherein the fragment of the Ig constant region includes a CH3 domain. quality.

55. Claim 52, wherein the fragment of the Ig constant region includes a CH2 domain and a CH3 domain. The isolated protein described.

56. The fragment of the Ig constant region comprises at least a part of the hinge, the CH2 domain, and the CH3 domain. The isolated protein according to claim 55, including the main protein.

57. The fragment of the Ig constant region includes a hinge, a CH2 domain, and a CH3 domain. The isolated protein described in item 56.

58. The antigen-binding domain that binds to hK2 is the Ig constant region or the Ig constant region The isolation agent according to any one of claims 50 to 57, which is conjugated at the N-terminus of the fragment. Protein.

59. The antigen-binding domain that binds to hK2 is the Ig constant region or the Ig constant region The isolation agent according to any one of claims 50 to 57, which is conjugated at the C-terminus of the fragment. Protein.

60. The antigen-binding domain that binds to hK2 is transmitted to Ig via the second linker (L2). Conjugated to a steady-state region or a fragment of the Ig steady-state region, any of claims 50 to 57 The isolated protein described in one of the items.

61. The aforementioned L2 corresponds to sequence numbers 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, ８５、８６、８７、８８、８９、９０、９１、９２、９３、９４、９５、９６、９７、９ 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 1 The isolated protein according to claim 60, comprising the amino acid sequence 08.

62. The Ig steady-state region or a fragment of the Ig steady-state region is IgG1, IgG2, IgG3, or The isolated protein according to any one of claims 51 to 61 is an IgG4 isotype. quality.

63. The Ig steady-state region or a fragment of the Ig steady-state region is IgG1, as described in claim 62. Isolated protein.

64. The Ig constant region or a fragment of the Ig constant region is the Fcγ receptor (F) of the protein. Any one of claims 51 to 63, comprising at least one mutation that reduces binding to cγR. The isolated protein described in item 1.

65. The at least one mutation that reduces the binding of the protein to the FcγR is F2 34A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331 S, F234A / L235A, S228P / F234A / L235A, N297A, V2 34A / G237A, K214T / E233P / L234V / L235A / G236 missing / A327G / P331A / D365E / L358M, H268Q / V309L / A33 0S / P331S, S267E / L328F, L234F / L235E / D265A, L 234A / L235A / G237A / P238S / H268A / A330S / P331S , S228P / F234A / L235A / G237A / P238S, and S228P / F Selected from the group consisting of 234A / L235A / G236 deletion / G237A / P238S The isolated protein according to claim 64, wherein the numbering of residues follows the EU index.

66. The mutation that reduces the binding of the protein to the FcγR is L234A_L235 The isolated protein according to claim 65, which is A_D265S.

67. The Ig constant region or a fragment of the Ig constant region of the protein to the FcγR The isolated protein according to claims 51 to 66, comprising at least one mutation that enhances binding. 。

68. The at least one mutation that enhances the binding of the protein to the FcγR is S2 39D / I332E, S298A / E333A / K334A, F243L / R292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292 P / Y300L / V305I / P396L, and G236A / S239D / I332E The isolation according to claim 67, selected from the group and the numbering of residues according to the EU index. protein.

69. The FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRI II, or any combination thereof, as described in any one of claims 64 to 68 Isolated protein.

70. The Ig constant region or a fragment of the Ig constant region regulates the half-life of the protein. The isolated protein according to any one of claims 51 to 63, comprising at least one mutation 。

71. At least one mutation that regulates the half-life of the aforementioned protein is H435A, P257I / N434H, D376V / N434H, M252Y / S254T / T256E / H43 Selected from the group consisting of 3K / N434F, T308P / N434A, and H435R, The isolated protein according to claim 70, wherein the numbering of residues follows the EU index.

72. Claims 51 to 63, comprising at least one mutation in the CH3 domain of the Ig constant region. The isolated protein described in any one of the items.

73. The at least one mutation in the CH3 domain of the Ig constant region is T35 0V, L351Y, F405A, Y407V, T366Y, T366W, F405W, T 394W, T394S, Y407T, Y407A, T366S / L368A / Y407V , L351Y / F405A / Y407V, T366I / K392M / T394W, F40 5A / Y407V, T366L / K392M / T394W, L351Y / Y407A, T 366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407V, and T350V / T Selected from the group consisting of 366L / K392L / T394W, the residue numbering is EU indexed. The isolated protein according to claim 72, conforming to the cues.

74. The mutation in the CH3 domain of the Ig constant region is T350V_T366L It is _K392L_T394W or T350V_L351Y_F405A_Y407V The isolated protein according to claim 73.

75. Claims that the IgG steady-state region includes SEQ ID NOs: 109, 110, 158, or 378. The isolated protein described in 73 or 74.

76. The isolation group according to claim 75, wherein the IgG steady-state region includes sequence number 109 or 378. Protein.

77. The isolated protein according to any one of claims 1 to 18 or 50 to 76, including the following: a. The heavy chain (HC) of SEQ ID NO: 210 and the light chain (LC) of SEQ ID NO: 221; b. HC of SEQ ID NO: 211 and LC of SEQ ID NO: 222; c. HC of SEQ ID NO: 212 and LC of SEQ ID NO: 223; d. HC of SEQ ID NO: 213 and LC of SEQ ID NO: 224; or e. HC of SEQ ID NO: 219 and LC of SEQ ID NO:

220.

78. Sequence ID 354 and at least 80% (for example, at least 85%, at least 90%) HC and Sequence ID No. 22 are identical (at least 95%, at least 99%, or 100%). 1 and at least 80% (for example, at least 85%, at least 90%, at least 95%) Any of claims 1 to 76, comprising LC that is identical by %, at least 99%, or 100%. The isolated protein described in the document.

79. An isolated protein containing an antigen-binding domain that binds to hK2, wherein the antigen-binding domain The isolated protein contains the following: a. HCDs of sequence numbers 170, 171, 172, 173, 174, and 175, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and / or teeth b. VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; and / or c. HC of SEQ ID NO: 210 and LC of SEQ ID NO:

221.

80. An isolated protein containing an antigen-binding domain that binds to hK2, wherein the antigen-binding domain The isolated protein contains the following: a. HCDs of sequence numbers 170, 171, 172, 173, 174, and 175, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and / or teeth b. VH of SEQ ID NO: 162 and VL of SEQ ID NO: 163; and / or c. HC of SEQ ID NO: 354 and LC of SEQ ID NO:

221.

81. An isolated protein containing an antigen-binding domain that binds to hK2, wherein the antigen-binding domain The isolated protein contains the following: a. HCDs of sequence numbers 176, 177, 178, 179, 180, and 181, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and / or teeth b. VH of sequence number 164 and VL of sequence number 165; and / or c. HC of SEQ ID NO: 211 and LC of SEQ ID NO:

222.

82. An isolated protein containing an antigen-binding domain that binds to hK2, wherein the antigen-binding domain The isolated protein contains the following: a. HCDs of sequence numbers 170, 183, 184, 185, 186, and 187, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and / or teeth b. VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167; and / or c. HC of SEQ ID NO: 212 and LC of SEQ ID NO:

223.

83. An isolated protein containing an antigen-binding domain that binds to hK2, wherein the antigen-binding domain The isolated protein contains the following: a. HCDs of sequence numbers 188, 189, 190, 191, 192, and 193, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and / or teeth b. VH of SEQ ID NO: 168 and VL of SEQ ID NO: 169; and / or c. HC of SEQ ID NO: 213 and LC of SEQ ID NO:

224.

84. An isolated protein containing an antigen-binding domain that binds to hK2, wherein the antigen-binding domain The isolated protein contains the following: a. HCDs of sequence numbers 206, 207, 208, 182, 470, and 209, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; and / or teeth b. VH of SEQ ID NO: 204 and VL of SEQ ID NO: 205; and / or c. HC of SEQ ID NO: 219 and LC of SEQ ID NO:

220.

85. The isolated cytoplasm according to any one of claims 79 to 84, which is a chimeric antigen receptor (CAR). Protein.

86. Claim 79 is a multispecific protein containing an antigen-binding domain that binds to CD3ε. Isolated protein as described in any one of items ~84.

87. The multiplicity feature according to claim 86, wherein the antigen-binding domain that binds to the CD3ε includes the following: Isogamous proteins: a. Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 116, HCD of SEQ ID NO: 117 R2, HCDR3 of SEQ ID NO: 118, Light chain complementarity determination region 1 (LCDR of SEQ ID NO: 119) 1) LCDR2 of SEQ ID NO: 120, and LCDR3 of SEQ ID NO: 121; or b. VH of sequence number 122 and VL of sequence number 123.

88. The antigen-binding domains that bind to the aforementioned CD3ε are VH of SEQ ID NO: 248 and SEQ ID NO: 15 The multispecific protein according to claim 86 or 87, comprising VL 7.

89. The antigen-binding domain that binds to the CD3ε comprises the following, as described in claim 86 or 87. multispecific proteins: a. Heavy chain complementarity determination region 1 (HCDR1) of SEQ ID NO: 255, HCD of SEQ ID NO: 256 R2, HCDR3 of SEQ ID NO: 257, light chain complementarity determination region 1 (LCDR of SEQ ID NO: 258) 1) LCDR2 of SEQ ID NO: 259, and LCDR3 of SEQ ID NO: 260; or b. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, H of SEQ ID NO: 257 CDR3, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and SEQ ID NO: 2 61 LCDR3; or c. VH of sequence number 248 and VL of sequence number 249; or d. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 250; or e. VH of sequence number 248 and VL of sequence number 251; or f. VH of sequence number 248 and VL of sequence number 252; or g. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 253; or h. VH of sequence number 248 and VL of sequence number 254.

90. The antigen-binding domain that binds to the aforementioned CD3ε is HCDR1 of SEQ ID NO. 255, SEQ ID NO. HCDR2 at sequence number 256, HCDR3 at sequence number 257, LCDR1 at sequence number 258, sequence The claim 86 includes LCDR2 number 259 and LCDR3 number 261. Multiple specificity proteins.

91. The antigen-binding domain that binds to the CD3ε is at least 80% of the VH of Sequence ID No.

248. % (for example, at least 85%, at least 90%, at least 95%, at least 99%) VH and VL of Sequence ID No. 251 are identical in % or 100% of the original (e.g., at least 80%) If so, at least 85%, at least 90%, at least 95%, at least 99%, or The multispecific protein according to claim 86 or 90, comprising VL which is 100% identical.

92. An isolated tranectomycete according to any one of claims 1 to 32, which is a chimeric antigen receptor (CAR). Pak quality.

93. The isolated protein according to claim 92, wherein the CAR comprises the following a. comprising an antigen-binding domain that binds to hK2 as described in any one of claims 1 to 32 Extracellular domain and b. Transmembrane domain and c. An intracellular signaling domain optionally containing at least one co-stimulatory domain.

94. The isolation tank according to claim 92 or 93, wherein the CAR further includes the CD8a hinge region. Pak quality.

95. The isolated protein according to any one of claims 92 to 94 is as follows: a. The CD8a hinge region is at least 90% identical to the amino acid sequence of Sequence ID No.

25. It contains the amino acid sequence; b. The transmembrane domain is at least 90% identical to the amino acid sequence of SEQ ID NO:

26. It includes an amino acid sequence; and / or c. The intracellular signaling domain has at least 9 amino acids from the amino acid sequence of Sequence ID No.

27. The co-stimulatory domain having an amino acid sequence that is 0% identical and the amino acid sequence of SEQ ID NO: 28 It contains a primary signaling domain having an amino acid sequence that is at least 90% identical.

96. The first antigen-binding domain binds to hK2 and the second antigen-binding domain binds to lymphocyte antigens. Isolated anti-hK2 / anti-CD3 protein containing the domain.

97. The isolated anti-hK2 / anti-CD3 according to claim 96, wherein the lymphocyte antigen is a T cell antigen. protein.

98. The T cell antigen is CD8 ＋ The isolated anti-hK2 / anti-T cell antigen according to claim 97. CD3 protein.

99. The isolated anti-hK2 / anti-CD antigen according to claim 98, wherein the lymphocyte antigen is an NK cell antigen.

3. Protein.

100. The lymphocyte antigens are CD3, CD3 epsilon (CD3ε), CD8, KI2L4, NKG2E, NKG2D, NKG2F, BTNL3, CD186, BTNL8, PD-1 The isolated antibody according to any one of claims 96 to 99, which is CD195 or NKG2C. hK2 / anti-CD3 protein.

101. The isolated anti-hK2 / anti-CD3 according to claim 100, wherein the lymphocyte antigen is CD3ε. protein.

102. The first antigen-binding domain that binds to the hK2 and / or the lymphocyte antigen. The second antigen-binding domain is scFv, (scFv) ２ , Fv, Fab, F(ab') ２ The isolated antibody according to any one of claims 96 to 100, comprising Fd, dAb, or VHH hK2 / anti-CD3 protein.

103. The first antigen-binding domain that binds to the hK2 and / or the lymphocyte antigen. The isolated anti-hK2 / anti-CD compound according to claim 102, wherein the second antigen-binding domain comprises Fab.

3. Protein.

104. The first antigen-binding domain that binds to the hK2 and / or the lymphocyte antigen. The isolated anti-hK2 / anti-CD2 according to claim 102, wherein the second antigen-binding domain comprises VHH.

3. Protein.

105. The first antigen-binding domain that binds to the hK2 and / or the lymphocyte antigen. The isolated anti-hK2 / anti-C2 according to claim 102, wherein the second antigen-binding domain comprises scFV. D3 protein.

106. The first antigen-binding domain that binds to hK2 contains Fab, and binds to the lymphocyte antigen. The isolated anti-hK2 / Anti-CD3 protein.

107. The scFv is VH, the first linker (L1), and from the N-terminus to the C-terminus, VL (VH-L1-VL), or including VL, L1, and VH (VL-L1-VH), The isolated anti-hK2 / anti-CD3 protein described in item 105 or 106.

108. The isolated anti-hK2 / anti-CD3 protein according to claim 107, wherein L1 comprises the following: a. Approximately 5 to 50 amino acids, b. Approximately 5 to 40 amino acids, c. Approximately 10 to 30 amino acids, or d. Approximately 10 to 20 amino acids.

109. The aforementioned L1 corresponds to sequence numbers 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, ８５、８６、８７、８８、８９、９０、９１、９２、９３、９４、９５、９６、９７、９ 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 1 The isolated anti-hK2 / anti-CD3 protein according to claim 108, comprising the amino acid sequence 08.

110. The isolated anti-hK2 / according to claim 109, wherein L1 includes the amino acid sequence of SEQ ID NO: 7 Anti-CD3 protein.

111. The first antigen-binding domain that binds to the aforementioned hK2 is sequence numbers 111, 112, 113, Claims 96-1, which specifically bind to the epitope on hK2 described in 114 or 115. The isolated anti-hK2 / anti-CD3 protein described in any one of the ten items.

112. The first antigen-binding domain that binds to the hK2 is described in Sequence IDs 111 and 112. The isolated anti-hK2 / anti-CD according to claim 111, which specifically binds to an epitope on hK2.

3. Protein.

113. The first antigen-binding domain that binds to the aforementioned hK2 is sequence numbers 111, 112, 113, hK2 containing one or more amino acid sequences selected from the group consisting of 114 and 115 The isolated anti-hK2 / anti-hK2 according to claim 111 or 112, which specifically binds to a continuous epitope. CD3 protein.

114. The second antigen-binding domain that binds to the CD3ε is sequence number 341, 448, or 4 Any of claims 96 to 113, which specifically binds to the epitope on CD3ε described in 49. The isolated anti-hK2 / anti-CD3 protein described in item 1.

115. The second antigen-binding domain that binds to the aforementioned CD3ε is sequence numbers 341, 448, and 4 Discontinuous epithet on CD3ε containing one or more amino acid sequences selected from a group of 49 The isolated anti-hK2 / anti-CD3 protein according to claim 114, which specifically binds to the rhombic linkage.

116. The second antigen-binding domain that binds to the aforementioned CD3ε is sequence numbers 341, 448, and 4 The isolated antibody according to claim 115, which specifically binds to the epitope on CD3ε described in 49. hK2 / anti-CD3 protein.

117. The first antigen-binding domain that binds to the aforementioned hK2 is sequence numbers 63, 72, 141, and 14. 7, 170, 176, 188, 194, 196, 198, 200, 206, or 216 HCDR1, SEQ ID NOs: 64, 65, 73, 142, 148, 171, 177, 189, 1 HCDR2 95, 197, 199, 201, 207, or 217, SEQ ID NOs: 66, 14 HCDR3 3, 172, 178, 184, 190, 208, or 218, SEQ ID NO: 67 , LCDR1 of 68, 144, 173, 179, 182, 185, or 191, Sequence ID LCDR2 69, 70, 145, 174, 180, 186, 192, or 470, and Includes LCDR3 of sequence numbers 71, 146, 175, 181, 187, 193, or 209. The isolated anti-hK2 / anti-CD3 protein according to any one of claims 96 to 116.

118. The first antigen-binding domain that binds to the aforementioned hK2 is one of the following: HCDR1, HCDR2, H The isolation according to claim 117, comprising CDR3, LCDR1, LCDR2, and LCDR3. anti-hK2 / anti-CD3 protein a. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; b. Sequence IDs 170, 171, 172, 173, 174, and 175, respectively; c. Sequence IDs 176, 177, 178, 179, 180, and 181, respectively; d. Sequence IDs 170, 183, 184, 185, 186, and 187, respectively; e. Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; f. Sequence numbers 206, 207, 208, 182, 470, and 209, respectively; g. Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; h. Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; i. Sequence numbers 196, 197, 178, 179, 190, and 181, respectively; j. Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; k. Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or l. Sequence numbers 216, 217, 218, 181, 470, and 209, respectively.

119. The first antigen-binding domain that binds to the aforementioned hK2 is sequence number 170, 171, HCDR1, HCDR2, HCDR3, LCDR 172, 173, 174, and 175 1. Isolated anti-hK2 / anti-CD3 according to claim 118, comprising LCDR2 and LCDR3. protein.

120. The first antigen-binding domain that binds to hK2 comprises the following, according to claim 118. Isolated anti-hK2 / anti-CD3 protein: a. VH of sequence number 137 and VL of sequence number 138; b. VH of sequence number 162 and VL of sequence number 163; c. VH of sequence number 164 and VL of sequence number 165; d. VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167; e. VH of sequence number 168 and VL of sequence number 169; f. VH of sequence number 204 and VL of sequence number 205; or g. VH of sequence number 166 and VL of sequence number 444.

121. The first antigen-binding domain that binds to hK2 is at least VH of SEQ ID NO: 162 80% (for example, at least 85%, at least 90%, at least 95%, at least) VH and VL of SEQ ID NO: 163 are identical by 99% or 100% and at least 80% ( For example, at least 85%, at least 90%, at least 95%, at least 99%, A single unit according to any one of claims 117 to 119, which includes a VL that is identical (or 100%). Anti-hK2 / anti-CD3 protein.

122. The first antigen-binding domain that binds to the aforementioned hK2 is VH of SEQ ID NO: 75 and SEQ ID NO: 7 The isolated anti-hK2 / anti-CD3 tadpole according to any one of claims 96 to 115, comprising 4 VL. Protein.

123. The first antigen-binding domain that binds to the aforementioned hK2 is sequence numbers 4, 5, 6, 137, 13 Includes VH of 9, 159, or 161 and VL of sequence numbers 1, 2, 3, 140, or 160. However, the antigen-binding domain that binds to hK2 is VH of SEQ ID NO: 5 and SEQ ID NO: The isolated anti-hK2 / anti-CD3 protein according to claim 122, which does not contain both of the two VLs. Quality.

124. The first antigen-binding domain that binds to the hK2 comprises the following, according to claim 123. Isolated anti-hK2 / anti-CD3 protein: a. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1; b. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2; c. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3; d. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; e. VH of sequence number 4 and VL of sequence number 160; f. VH of sequence number 5 and VL of sequence number 1; g. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; h. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; i. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; j. VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1; k. VH of sequence number 6 and VL of sequence number 2; l. VH of sequence number 6 and VL of sequence number 3; m. VH of sequence number 6 and VL of sequence number 140; n. VH of sequence number 6 and VL of sequence number 160; o. VH of sequence number 139 and VL of sequence number 1; p. VH of sequence number 139 and VL of sequence number 2; q. VH of sequence number 139 and VL of sequence number 3; r. VH of sequence number 139 and VL of sequence number 140; s. VH of sequence number 139 and VL of sequence number 160; t. VH of sequence number 159 and VL of sequence number 1; u. VH of sequence number 159 and VL of sequence number 2; v. VH of sequence number 159 and VL of sequence number 3; w. VH of sequence number 159 and VL of sequence number 140; x. VH of sequence number 159 and VL of sequence number 160; y. VH of sequence number 161 and VL of sequence number 1; z. VH of sequence number 161 and VL of sequence number 2; aa. VH of sequence number 161 and VL of sequence number 3; bb. VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or cc. VH of sequence number 161 and VL of sequence number 160.

125. The first antigen-binding domain that binds to hK2 is sequence numbers 8, 9, 10, 11, 12 、１３、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、１３３、１３ ４、１３５、１３６、３０８、３１６、３１８、３１９、３２０、３２１、３２２、３２ 3, 324, 325, 404, 405, 406, 407, 408, or 409 amino acids The isolated anti-hK2 / anti-CD3 protein according to claim 124, comprising a sequence.

126. The first antigen-binding domain that binds to hK2 is the amino acid of sequence number 404 or 405. Acid sequence and at least 80% (e.g., at least 85%, at least 90%, at least Claim 12, comprising an amino acid sequence that is 95%, at least 99%, or 100% identical. The isolated protein described in 4.

127. The second antigen-binding domain that binds to the lymphocyte antigen is sequence number 255 or 116. HCDR1, HCDR2 of SEQ ID NO: 256 or 117, HCDR2 of SEQ ID NO: 257 or 118 DR3, LCDR1 of sequence number 258 or 119, LCDR of sequence number 259 or 120 Claims 96 to 125, which include 2 and LCDR3 of sequence number 260, 261, or 121. The isolated anti-hK2 / anti-CD3 protein described in any one of the items.

128. The second antigen-binding domain that binds to the lymphocyte antigen is HCDR1 of SEQ ID NO: 255 , HCDR2 of SEQ ID NO: 256, HCDR3 of SEQ ID NO: 257, LCD of SEQ ID NO: 258 Claim 1, comprising R1, LCDR2 of Sequence ID No. 259, and LCDR3 of Sequence ID No.

261. The isolated anti-hK2 / anti-CD3 protein described in 26.

129. The second antigen-binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 248 and The isolated anti-hK2 / anti-CD3 tadpole according to claim 126 or 127, comprising VL of row number 157. Protein.

130. Claim 128 or the second antigen-binding domain that binds to the lymphocyte antigen comprises the following: This is the isolated anti-hK2 / anti-CD3 protein described in 129. a. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, H of SEQ ID NO: 257 CDR3, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and SEQ ID NO: 2 60 LCDR3; b. HCDR1 of SEQ ID NO: 255, HCDR2 of SEQ ID NO: 256, H of SEQ ID NO: 257 CDR3, LCDR1 of SEQ ID NO: 258, LCDR2 of SEQ ID NO: 259, and SEQ ID NO: 2 61 LCDR3; c. VH of sequence number 248 and VL of sequence number 249; d. VH of sequence number 248 and VL of sequence number 250; e. VH of sequence number 248 and VL of sequence number 251; f. VH of sequence number 248 and VL of sequence number 252; g. VH of SEQ ID NO: 248 and VL of SEQ ID NO: 253; or h. VH of sequence number 248 and VL of sequence number 254.

131. The second antigen-binding domain that binds to the lymphocyte antigen is less than that of VH in SEQ ID NO:

248. At least 80% (for example, at least 85%, at least 90%, at least 95%, less VH and VL of Sequence ID No. 251 are identical by at least 99% or 100%. 0% (for example, at least 85%, at least 90%, at least 95%, at least 9 The isolated anti-hK2 / anti-C compound according to claim 129, comprising 9% or 100% identical VL. D3 protein.

132. Claims 96-1, wherein the second antigen-binding domain that binds to the lymphocyte antigen includes the following: Isolated anti-hK2 / anti-CD3 protein as described in any one of item 26 a. HCDR1 of SEQ ID NO: 116, HCDR2 of SEQ ID NO: 117, H of SEQ ID NO: 118 CDR3, LCDR1 of SEQ ID NO: 119, LCDR2 of SEQ ID NO: 120, and SEQ ID NO: 1 21 LCDR3; or b. VH of sequence number 122 and VL of sequence number 123.

133. The second antigen-binding domain that binds to the lymphocyte antigen is sequence numbers 328, 329, 3 The amino acid sequence 30, 331, 332, 333, 334, 335, 336, or 337 Including the isolated anti-hK2 / anti-CD3 protein according to claim 132.

134. The second antigen-binding domain that binds to the lymphocyte antigen is the amino acid sequence of SEQ ID NO:

331. Columns and at least 80% (e.g., at least 85%, at least 90%, at least 95%) Claims 96 to 1 include an amino acid sequence that is identical by %), at least 99%, or 100%. The isolated anti-hK2 / anti-CD3 protein described in 32.

135. The first antigen-binding domain that binds to hK2 is the first immunoglobulin (Ig) constant Conjugated to a region or a fragment of the first Ig constant region, and / or the lymph The second antigen-binding domain, which binds to the globular antigen, is located in the second immunoglobulin (Ig) constant region. or conjugate to a fragment of the second Ig steady-state region, any one of claims 96 to 134 The isolated anti-hK2 / anti-CD3 protein described in item 1.

136. A first antigen-binding domain that binds to the hK2 and the first Ig constant region or the first Between the fragment of the Ig constant region and the second antigen-binding domain that binds to the lymphocyte antigen. A second linker is placed between the second Ig steady-state region or a fragment of the second Ig steady-state region. The isolated anti-hK2 / anti-CD3 protein according to claim 135, further comprising (L2).

137. The aforementioned L2 corresponds to sequence numbers 7, 76, 77, 78, 79, 80, 81, 82, 83, 84, ８５、８６、８７、８８、８９、９０、９１、９２、９３、９４、９５、９６、９７、９ 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, or 1 The isolated anti-hK2 / anti-CD3 protein according to claim 136, comprising the amino acid sequence 08.

138. The isolated anti-hK2 / anti-hK2 / anti-hK2 according to claim 137, wherein the fragment of the Ig steady-state region includes an Fc region. CD3 protein.

139. The isolated anti-hK according to claim 138, wherein the fragment of the Ig constant region includes a CH2 domain. 2 / Anti-CD3 protein.

140. The isolated antihK according to claim 139, wherein the fragment of the Ig constant region includes a CH3 domain. 2 / Anti-CD3 protein.

141. Claim 140, wherein the fragment of the Ig constant region includes a CH2 domain and a CH3 domain. The isolated anti-hK2 / anti-CD3 protein described above.

142. The fragment of the Ig constant region comprises at least a part of the hinge, the CH2 domain, and the CH3 domain. The isolated anti-hK2 / anti-CD3 protein according to claim 141, including the main protein.

143. The fragment of the Ig constant region includes a hinge, a CH2 domain, and a CH3 domain. The isolated anti-hK2 / anti-CD3 protein described in item 142.

144. The first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region The region or the fragment of the second Ig steady-state region is IgG1, IgG2, and IgG3, or I The isolated anti-hK2 / anti-CD3 protein according to claim 138, which is the γ4 isotype.

145. The first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region The isolated antibody according to claim 144, wherein the region or the fragment of the second Ig constant region is IgG1. hK2 / anti-CD3 protein.

146. The first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region The region or the fragment of the second Ig constant region binds the multispecific protein to FcγR. The isolated anti-hK2 / anti-CD compound according to claim 145, comprising at least one mutation that reduces the compound.

3. Protein.

147. The at least one mutation that reduces the binding of the multispecific protein to the FcγR The differences are F234A / L235A, L234A / L235A, L234A / L235A / D 265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A / L235A, N29 7A, V234A / G237A, K214T / E233P / L234V / L235A / G 236 Missing / A327G / P331A / D365E / L358M, H268Q / V309 L / A330S / P331S, S267E / L328F, L234F / L235E / D2 65A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S2 Is it a group consisting of 28P / F234A / L235A / G236 (deleted) / G237A / P238S? The isolated anti-hK2 according to claim 146, selected and whose residue numbering follows the EU index. / Anti-CD3 protein.

148. The mutation that reduces the binding of the multispecific protein to the FcγR is L234A_L The isolated anti-hK2 / anti-CD3 protein according to claim 147 is 235A_D265S. 。

149. The first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region The region or the fragment of the second Ig constant region is the Fcγ receptor of the multispecific protein ( The isolation according to claim 144, comprising at least one mutation that enhances binding to FcγR). Anti-hK2 / anti-CD3 protein.

150. The at least one mutation that enhances the binding of the multispecific protein to the FcγR The difference is S239D / I332E, S298A / E333A / K334A, F243L / R 292P / Y300L, F243L / R292P / Y300L / P396L, F243L / R292P / Y300L / V305I / P396L, and G236A / S239D / I Claim 149, selected from the group consisting of 332E, wherein the numbering of residues follows the EU index. The isolated anti-hK2 / anti-CD3 protein described above.

151. The FcγR is FcγRI, FcγRIIA, FcγRIIB, or FcγRI II, or any combination thereof, as described in any one of claims 146 to 150 The isolated anti-hK2 / anti-CD3 protein shown.

152. The first Ig steady-state region or a fragment of the first Ig steady-state region, and the second Ig steady-state region or A second Ig constant region fragment modulates at least one of the half-lives of multispecific proteins. The isolated multispecific protein according to claim 144, comprising the mutation.

153. The at least one mutation that regulates the half-life of the multispecific protein is H4 35A, P257I / N434H, D376V / N434H, M252Y / S254T / From T256E / H433K / N434F, T308P / N434A, and H435R The isolated antibody according to claim 152, selected from the group and having residue numbering according to the EU index hK2 / anti-CD3 protein.

154. C of the CH3 domain or fragment of the first Ig steady-state region At least one mutation in the H3 domain and / or the CH3 of the second Ig constant region At least one CH3 domain of the domain or the fragment of the second Ig constant region The isolated anti-hK2 / anti-CD according to either claim 152 or 153, comprising one mutation.

3. Protein.

155. C of the CH3 domain or fragment of the first Ig steady-state region The at least one mutation in the H3 domain and / or the C in the second Ig constant region In the H3 domain or the CH3 domain of the fragment of the second Ig constant region, Another mutation is T350V, L351Y, F405A, Y407V, T366Y, T3 66W, F405W, T394W, T394S, Y407T, Y407A, T366S / L368A / Y407V, L351Y / F405A / Y407V, T366I / K392 M / T394W, F405A / Y407V, T366L / K392M / T394W, L3 51Y / Y407A, T366A / K409F, L351Y / Y407A, T366V / K409F, T366A / K409F, T350V / L351Y / F405A / Y407 Selected from the group consisting of V and T350V / T366L / K392L / T394W, the remainder The isolated anti-hK2 / anti-CD3 tangent according to claim 154, wherein the base numbering follows the EU index. Pak quality.

156. CH3 domain of the first Ig steady-state region or CH3 of a fragment of the first Ig steady-state region The mutation in the domain and the CH3 domain or the second Ig constant region The mutations in the CH3 domain of the g constant region fragment are, respectively, T350V_T36 6L_K392L_T394W or T350V_L351Y_F405A_Y407V A certain isolated anti-hK2 / anti-CD3 protein according to claim 155.

157. The first Ig steady-state region or a fragment of the first Ig steady-state region and the second Ig steady-state region The region or the fragment of the second Ig constant region comprises the following mutations, according to claims 135 to 156. The isolated anti-hK2 / anti-CD3 protein described in either item. a. L234A_L235A_D265S_T350 in the first steady-state Ig region V_L351Y_F405A_Y407V and L234 in the second Ig steady-state region A_L235A_D265S_T350V_T366L_K392L_T394W; or b. L234A_L235A_D265S_T350 in the first steady-state Ig region V_T366L_K392L_T394W and L234 in the second Ig steady-state region A_L235A_D265S_T350V_L351Y_F405A_Y407V.

158. Claim 15, wherein the first Ig steady-state region has sequence number 110 or sequence number 378. The isolated anti-hK2 / anti-CD3 protein described in 7.

159. Claim 15, wherein the second Ig steady-state region has sequence numbers 109 and 158. The isolated anti-hK2 / anti-CD3 protein described in either item 7 or 158.

160. The first Ig steady-state region is at least 80% (for example, at least) of SEQ ID NO:

378. 85%, at least 90%, at least 95%, at least 99%, or 100%) identical The amino acid sequence includes the second Ig constant region, and the sequence number 109 and at least 8 0% (for example, at least 85%, at least 90%, at least 95%, at least 9 Any of claims 135 to 157, comprising an amino acid sequence that is identical by 9% or 100%. The isolated anti-hK2 / anti-CD3 protein described in item 1.

161. The isolated protein is at least 80% (for example, at least 85%) of SEQ ID NO:

309. %) identical The isolated anti-hK2 / anti-hK2 according to claim 160, further comprising a light chain constant region containing an amino acid sequence. CD3 protein.

162. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 63, 65, and 66, respectively. , 67, 69, and 71 HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2, and LCDR3, each comprising a second domain that binds to the lymphocyte antigen, HCDR1, HCDR2 in rows 255, 256, 275, 258, 259, and 261 , including HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 136, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 248 and SEQ ID NO: Including 251 VLs; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 351, H of SEQ ID NO: 358 Includes C2 and LC2 of Sequence ID No.

267.

163. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 63, 65, and 66, respectively. , 67, 69, and 71 HCDR1, HCDR2, HCDR3, LCDR1, LCDR 2, and LCDR3, each comprising a second domain that binds to the lymphocyte antigen, HCDR1 and HCDR2 in rows 116, 117, 118, 119, 120, and 121 , including HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 136, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 122 and SEQ ID NO: Including 123 VL; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 351, H of SEQ ID NO: 359 Includes C2 and LC2 of Sequence ID No.

272.

164. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 141 and 142, respectively. HCDR1, HCDR2, HCDR3, LCDR 143, 144, 145, and 146 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 134, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 248 and SEQ ID NO: Including 251 VLs; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 352, H of SEQ ID NO: 358 Includes C2 and LC2 of Sequence ID No.

267.

165. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 141 and 142, respectively. HCDR1, HCDR2, HCDR3, LCDR 143, 144, 145, and 146 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 116, 117, 118, 119, 120, and 121, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 134, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 122 and SEQ ID NO: Including 123 VL; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 352, H of SEQ ID NO: 359 Includes C2 and LC2 of Sequence ID No.

272.

166. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 188 and 189, respectively. HCDR1, HCDR2, HCDR3, LCDR 190, 191, 192, and 193 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 325, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 248 and SEQ ID NO: Including 251 VLs; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 353, H of SEQ ID NO: 358 Includes C2 and LC2 of Sequence ID No.

267.

167. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 170 and 171, respectively. HCDR1, HCDR2, HCDR3, LCDR 172, 173, 174, and 175 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first antigen-binding domain that binds to hK2 is VH and sequence of Sequence ID No.

162. The second antigen-binding domain, which includes the VL number 163 and binds to the lymphocyte antigen, is sequence Including scFV number 331; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 354, L of SEQ ID NO:

221. Includes C1 and HC2 of sequence number 360.

168. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 170 and 171, respectively. HCDR1, HCDR2, HCDR3, LCDR 172, 173, 174, and 175 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 is at least VH of SEQ ID NO: 162 80% (for example, at least 85%, at least 90%, at least 95%, at least) VH and VL of SEQ ID NO: 163 are identical by 99% or 100% and at least 80% ( For example, at least 85%, at least 90%, at least 95%, at least 99%, A second binding domain that binds to the lymphocyte antigen, comprising a VL that is identical (or 100%). However, the scFV of sequence number 331 is at least 80% (for example, at least 85%, less Both are identical scFV (90%, at least 95%, at least 99%, or 100%). Including; and / or c. The isolated multispecific protein is at least 80% ( For example, at least 85%, at least 90%, at least 95%, at least 99%, HC1 is identical to LC1 of sequence number 221 (or 100%) and is at least 80% (for example) , at least 85%, at least 90%, at least 95%, at least 99%, or 1 LC1 is identical to 00%) and HC2 of Sequence ID No. 360 is at least 80% (for example, At least 85%, at least 90%, at least 95%, at least 99%, or 10 0%) Contains the same HC2.

169. The isolated multispecific proteins have lysine (for example, K4) at the C-terminus of HC1 and HC2. 77) comprising, the isolated multispecific protein is at least HC1 of SEQ ID NO: 361 80% (for example, at least 85%, at least 90%, at least 95%, at least) HC1 and HC2 of Sequence ID No. 362 are 99% or 100% identical, and at least 80 % (for example, at least 85%, at least 90%, at least 95%, at least 99%) The isolated anti-h2 according to claim 167 or 168, comprising HC2 that is identical in %) or 100%). K2 / anti-CD3 protein.

170. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 170 and 183, respectively. HCDR1, HCDR2, HCDR3, LCDR 184, 185, 186, and 187 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first antigen-binding domain that binds to hK2 is VH and sequence of Sequence ID No.

166. The VL containing number 444 has a second antigen-binding domain that binds to the lymphocyte antigen, Including scFV number 331; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 355, L of SEQ ID NO: 445 Includes C1 and HC2 of sequence number 360.

171. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 176 and 177, respectively. HCDR1, HCDR2, HCDR3, LCDR 178, 179, 180, and 181 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first antigen-binding domain that binds to hK2 is VH and sequence of Sequence ID No.

164. The second antigen-binding domain, which includes the VL number 165 and binds to the lymphocyte antigen, is sequence Including scFV number 331; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 356, L of SEQ ID NO: 222 Includes C1 and HC2 of sequence number 360.

172. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 188 and 189, respectively. HCDR1, HCDR2, HCDR3, LCDR 190, 191, 192, and 193 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 116, 117, 118, 119, 120, and 121, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 325, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 122 and SEQ ID NO: Including 123 VL; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 353, H of SEQ ID NO: 359 Includes C2 and LC2 of Sequence ID No.

272.

173. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 206, 207, respectively. 208, 182, 470, 209 HCDR1, HCDR2, HCDR3, LCDR1, The second domain, which includes LCDR2 and LCDR3 and binds to the lymphocyte antigen, HCDR1, H of sequence numbers 116, 117, 118, 119, 120, and 121, respectively. Including CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 316, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 122 and SEQ ID NO: Including 123 VL; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 357, H of SEQ ID NO: 359 Includes C2 and LC2 of Sequence ID No.

272.

174. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 206, 207, respectively. 208, 182, 470, 209 HCDR1, HCDR2, HCDR3, LCDR1, The second domain, which includes LCDR2 and LCDR3 and binds to the lymphocyte antigen, HCDR1, H of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. Including CDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first binding domain that binds to hK2 includes the scFV of sequence number 316, The second binding domain that binds to the lymphocyte antigen is VH of SEQ ID NO: 248 and SEQ ID NO: Including 151 VL; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 357, H of SEQ ID NO: 358 Includes C2 and LC2 of Sequence ID No.

267.

175. It includes a first domain that binds to hK2 and a second domain that binds to CD3, and below A certain isolated anti-hK2 / anti-CD3 protein a. The first binding domains that bind to hK2 are sequence numbers 170 and 171, respectively. HCDR1, HCDR2, HCDR3, LCDR 172, 173, 174, and 175 1. A second domain comprising LCDR2 and LCDR3 that binds to the lymphocyte antigen is HCDR1 of sequence numbers 255, 256, 257, 258, 259, and 261, respectively. , including HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3; b. The first antigen-binding domain that binds to hK2 is VH and sequence of Sequence ID No.

162. The second antigen-binding domain, which includes the VL number 163 and binds to the lymphocyte antigen, is sequence Including scFV number 331; and / or c. The isolated multispecific protein is HC1 of SEQ ID NO: 361, L of SEQ ID NO: 221 Includes C1 and HC2 of sequence number 362.

176. The isolation agent according to any one of claims 1 to 95, conjugated with a therapeutic agent or contrast agent. An immune conjugate containing protein.

177. An isolated protein according to any one of claims 1 to 95, and a pharmaceutically acceptable carrier A pharmaceutical composition containing the following:

178. Polynucleotide encoding an isolated protein according to any one of claims 1 to 95 Ochido.

179. It is a polynucleotide, a. Encoding an isolated protein according to any one of claims 1 to 95; and / or b. Sequence numbers 225, 226, 227, 228, 229, 230, 231, 232, 2 ３３、２３４、２３５、２３６、２３７、２３８、２３９、２４０、２４１、２４２、２ ４３、２４４、２４５、２４６、２４７、２６３、２７１、２７４、２８２、２９２、２ ９３、２９４、２９５、３０３、３０４、３０５、３０６、３１２、３１７、３６３、３ ６４、３６５、３６６、３６７、３６８、３６９、３７０、３７１、３７２、３７３、３ A polynucleotide containing a polynucleotide sequence of 74, 450, or 451.

180. It is a polynucleotide, a. The isolated protein according to any one of claims 1 to 95 and at least 80% (e.g. If so, at least 85%, at least 90%, at least 95%, at least 99%, or It codes for an isolated protein that is 100% identical; and / or b. Sequence numbers 225, 226, 227, 228, 229, 230, 231, 232, 2 ３３、２３４、２３５、２３６、２３７、２３８、２３９、２４０、２４１、２４２、２ ４３、２４４、２４５、２４６、２４７、２６３、２７１、２７３、２７４、２７５、２ ７６、２７７、２７８、２７９、２８０、２８１、２８２、２８３、２９２、２９３、２ ９４、２９５、２９６、２９７、３０３、３０４、３０５、３０６、３１２、３１６、３ １７、３６３、３６４、３６５、３６６、３６７、３６８、３６９、３７０、３７１、３ Polynucleotide sequences of 72, 373, 374, 446, 450, or 451 are small At least 80% (for example, at least 85%, at least 90%, at least 95%, less Polynucleotides containing polynucleotide sequences that are identical by at least 99% or 100% Do.

181. A vector comprising the polynucleotide described in claim 179 or 180.

182. A host cell comprising the vector according to claim 181.

183. A method for producing an isolated protein according to any one of claims 1 to 95, the The host cells described in claim 182 are cultured under conditions in which a protein is expressed, and the host A method comprising recovering the protein produced by the cell.

184. A single unit according to any one of claims 96 to 175, conjugated with a therapeutic agent or contrast agent. An immune conjugate containing a bispecific protein.

185. An isolated bispecific protein according to any one of claims 96 to 175, and a pharmaceutically acceptable A pharmaceutical composition comprising a carrier.

186. It is a polynucleotide, a. Encoding a bispecific protein according to any one of claims 96 to 175 ru; and / or b. Sequence numbers 225, 226, 227, 228, 229, 230, 231, 232, 2 ３３、２３４、２３５、２３６、２３７、２３８、２３９、２４０、２４１、２４２、２ ４３、２４４、２４５、２４６、２４７、２６３、２７１、２７４、２８２、２９２、２ ９３、２９４、２９５、３０３、３０４、３０５、３０６、３１２、３１６、３１７、３ ６３、３６４、３６５、３６６、３６７、３６８、３６９、３７０、３７１、３７２、３ Polynucleotide sequences containing 73, 374, 450, or 451 polynucleotides Do.

187. It is a polynucleotide, a. The isolated protein according to any one of claims 96 to 175 and at least 80% ( For example, at least 85%, at least 90%, at least 95%, at least 99%, Encoding an isolated protein that is identical (or 100%); and / or b. Sequence numbers 225, 226, 227, 228, 229, 230, 231, 232, 2 ３３、２３４、２３５、２３６、２３７、２３８、２３９、２４０、２４１、２４２、２ ４３、２４４、２４５、２４６、２４７、２６３、２７１、２７３、２７４、２７５、２ ７６、２７７、２７８、２７９、２８０、２８１、２８２、２８３、２９２、２９３、２ ９４、２９５、２９６、２９７、３０３、３０４、３０５、３０６、３１２、３１６、３ １７、３６３、３６４、３６５、３６６、３６７、３６８、３６９、３７０、３７１、３ Polynucleotide sequences of 72, 373, 374, 446, 450, or 451 are small At least 80% (for example, at least 85%, at least 90%, at least 95%, less Polynucleotides containing polynucleotide sequences that are identical by at least 99% or 100% Do.

188. A vector comprising the polynucleotide described in claim 186 or 187.

189. A host cell comprising the vector according to claim 188.

190. Method for generating an isolated bispecific protein according to any one of claims 96 to 175 The host cell according to claim 189 is expressed under conditions in which the multispecific protein is expressed. The process involves culturing the cells and recovering the multispecific proteins produced by the host cells. A method that includes the act of doing something.

191. A method for treating hK2-expressing cancer in a target, wherein the therapeutic effective amount is as described in claims 1 to 95. The isolated protein according to any one of claims, the isolated protein according to any one of claims 96 to 175 A heavy specific protein, an immunoconjugate according to claim 176 or 184, or The pharmaceutical composition described in item 177 or 185 is sufficient to treat the hK2-expressing cancer. A method comprising administering the substance to the subject over a period of time.

192. A method for reducing the amount of hK2-expressing tumor cells in a target, according to any of claims 1 to 95 The isolated protein according to any one of claims, the isolated double protein according to any one of claims 96 to 175 A specific protein, an immunoconjugate according to claim 176 or 184, or claim The pharmaceutical composition described in item 177 or 185 reduces the amount of the hK2-expressing tumor cells. A method comprising administering the substance to the subject for a sufficient period of time.

193. A method for preventing the engraftment of hK2-expressing cancer in a target, any one of claims 1 to 95. The isolated protein described in one claim, the isolated bispecific protein described in any one of claims 95 to 175 Sexual protein, immunoconjugate according to claim 176 or 184, or claim 1 The pharmaceutical composition described in 77 or 185 is administered to the subject, and the h in the subject A method including preventing the engraftment of K2-expressing cancer.

194. This was a method for treating non-cancerous conditions in subjects at risk of developing hK2-expressing cancer. The isolated protein according to any one of claims 1 to 95, or any of claims 96 to 175 The isolated multispecific protein described in any one of the claims, or the immunoassay described in claim 176 or 184. The conjugate, or the pharmaceutical composition according to claim 177 or 185, is administered to the subject. A method comprising treating the aforementioned non-cancerous condition.

195. Any one of claims 191 to 194, wherein the hK2-expressing cancer is prostate cancer or breast cancer. Methods used.

196. The aforementioned prostate cancer is recurrent, refractory, malignant, or castration-resistant prostate cancer, or these The method according to claim 195, which is any combination of the above.

197. The aforementioned breast cancer is androgen receptor (AR) positive breast cancer, recurrent, refractory, or malignant. The method according to claim 195, wherein breast cancer, or any combination thereof.

198. The aforementioned non-cancerous condition is benign prostatic hyperplasia, benign prostatic hyperplasia (BPH), or a diagnosed prostate. A condition in which there are high levels of prostate-specific antigen (PSA) in the absence of cancer, The method described in item 195.

199. The isolated protein or the isolated multispecific protein is combined with the second therapeutic agent. The method according to any one of claims 191 to 198, wherein the method is administered by [method].

200. The second therapeutic agent is used in conjunction with surgery, chemotherapy, androgen depletion therapy, or radiation therapy, or The method according to claim 199, which is any combination thereof.

201. A method for detecting the presence of prostate cancer or breast cancer in a subject, as described in claim 176 or 18. The immune conjugate described in 4 is administered to subjects suspected of having prostate cancer or breast cancer. This involves visualizing the biological structure to which the aforementioned immune conjugate is bound, and thereby A method comprising detecting the presence of prostate cancer or breast cancer.

202. The isolated protein according to any one of claims 1 to 95, or any one of claims 96 to 175. The isolated bispecific protein described in item 1, the immunoprotein described in claim 176 or 184 A kit comprising a jugate or the pharmaceutical composition described in claim 177 or 185.

203. Anti-idiotype that binds to the isolated protein according to any one of claims 1 to 95 antibody.

204. Chimeric antigen receptors (CARs), including the following: a. An extracellular domain containing an antigen-binding domain that binds to hK2, b. Transmembrane domain and c. An intracellular signaling domain optionally containing at least one co-stimulatory domain.

205. The CAR according to claim 204, further comprising a CD8a hinge region.

206. The CAR according to claim 204 or 205 is as follows: a. The transmembrane domain contains a CD8a transmembrane region (CD8A-TM) polypeptide. fruit; b. The intracellular signaling domain is a TNF receptor superfamily member 9 (CD137) component-containing co-stimulatory domain and T cell surface glycoprotein CD3 zeta chain ( It contains a primary signaling domain that includes the CD3z component.

207. The CAR according to claim 204 or 206 is as follows: a. The CD8a hinge region is at least 90% identical to the amino acid sequence of Sequence ID No.

25. It contains the amino acid sequence; b. The transmembrane domain is at least 90% identical to the amino acid sequence of SEQ ID NO:

26. It includes an amino acid sequence; and / or c. The intracellular signaling domain has at least 9 amino acids from the amino acid sequence of Sequence ID No.

27. The co-stimulatory domain having an amino acid sequence that is 0% identical and the amino acid sequence of SEQ ID NO: 28 It contains a primary signaling domain having an amino acid sequence that is at least 90% identical.

208. The antigen-binding domain that binds to the aforementioned hK2 is sequence numbers 63, 72, 141, 147, 1 HCDs of 70, 176, 188, 194, 196, 198, 200, 206, or 216 R1, Sequence IDs 64, 65, 73, 142, 148, 171, 177, 183, 189, HCDR2 195, 197, 199, 201, 207, or 217, SEQ ID NO: 66, 1 HCDR3 43, 172, 178, 184, 190, 208, or 218, Sequence ID 6 LCDR1 sequence numbers 7, 68, 144, 173, 179, 182, 185, or 191 LCDR2 numbers 69, 70, 145, 174, 180, 186, 192, or 470, and and LCDR3 with sequence numbers 71, 146, 175, 181, 187, 193, or 209 A CAR according to any one of claims 204 to 207.

209. The antigen-binding domain that binds to the aforementioned hK2 is one of the following: HCDR1, HCDR2, HCDR 3. Any of claims 204 to 208, including LCDR1, LCDR2, and LCDR3. CAR as described in item 1 a. Sequence IDs 63, 64, 66, 67, 69, and 71, respectively; b. Sequence IDs 63, 65, 66, 67, 69, and 71, respectively; c. Sequence IDs 63, 65, 66, 68, 70, and 71, respectively; d. Sequence numbers 141, 142, 143, 144, 145, and 146, respectively; e. Sequence IDs 170, 171, 172, 173, 174, and 175, respectively; f. Sequence numbers 176, 177, 178, 179, 180, and 181, respectively; g. Sequence IDs 170, 183, 184, 185, 186, and 187, respectively; h. Sequence numbers 188, 189, 190, 191, 192, and 193, respectively; i. Sequence numbers 206, 207, 208, 182, 470, or 209, respectively; j. Sequence IDs 72, 73, 66, 67, 69, and 71, respectively; k. Sequence IDs 72, 73, 66, 68, 70, and 71, respectively; l. Sequence numbers 147, 148, 143, 144, 145, and 146, respectively; m. Sequence numbers 194, 195, 172, 173, 174, and 175, respectively; n. Sequence numbers 196, 197, 178, 179, 180, and 181, respectively; o. Sequence numbers 198, 199, 184, 185, 186, and 187, respectively; p. Sequence numbers 200, 201, 190, 191, 192, and 193, respectively; or q. Sequence numbers 216, 217, 218, 182, 470, and 209, respectively.

210. The antigen-binding domain that binds to hK2 is LCDR1 of VL in Sequence ID No. 163, LC DR2, and LCDR3, and HCDR1, HCDR2, and VH of Sequence ID No. 162 A CAR according to any one of claims 204 to 209, comprising HCDR3.

211. A CAR according to any one of claims 204 to 209, wherein the hK2 is bonded The antigen-binding domains are LCDR1, LCDR2, and LCDR3 of the VL of SEQ ID NO: 138 , and including HCDR1, HCDR2, and HCDR3 of VH in Sequence ID No. 137; or LCDR1, LCDR2, and LCDR3 of VL in Sequence ID No. 140, and Sequence ID No. 13 A CAR comprising HCDR1, HCDR2, and HCDR3 of VH 9.

212. The antigen-binding domain that binds to the hK2 comprises the following, according to claims 204 to 211 CAR as described in any one of the items a. VH of sequence number 137 and VL of sequence number 138; b. VH of sequence number 162 and VL of sequence number 163; c. VH of sequence number 164 and VL of sequence number 165; d. VH of SEQ ID NO: 166 and VL of SEQ ID NO: 167; e. VH of sequence number 168 and VL of sequence number 169; f. VH of sequence number 204 and VL of sequence number 205; or g. VH of sequence number 166 and VL of sequence number 444.

213. The antigen-binding domain that binds to the aforementioned hK2 is VH in SEQ ID NO: 75 and V in SEQ ID NO:

74. A CAR according to any one of claims 204 to 212, including L.

214. The antigen-binding domain that binds to hK2 is the amine of SEQ ID NO: 138 or SEQ ID NO:

140. No acid sequence and at least 80% (e.g., at least 85%, at least 90%, at least) VL and compound containing amino acid sequences that are identical (95%, at least 99%, or 100%) The amino acid sequence of sequence number 137 or sequence number 139 and at least 80% (for example, at least (85%, at least 90%, at least 95%, at least 99%, or 100%) The VH comprising a single amino acid sequence, according to any one of claims 204 to 211. CAR.

215. The antigen-binding domain that binds to the aforementioned hK2 is sequence number 1, 2, 3, 140, or 1 Includes VL 60 and / or VH of sequence numbers 4, 5, 6, 139, 159, or 161 The CAR described in claim 213.

216. The CAR according to claim 215, comprising the antigen-binding domain that binds to the hK2, ： a. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 1; b. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 2; c. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 3; d. VH of SEQ ID NO: 4 and VL of SEQ ID NO: 140; e. VH of sequence number 4 and VL of sequence number 160; f. VH of sequence number 5 and VL of sequence number 1; g. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 3; h. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 140; i. VH of SEQ ID NO: 5 and VL of SEQ ID NO: 160; j. VH of SEQ ID NO: 6 and VL of SEQ ID NO: 1; k. VH of sequence number 6 and VL of sequence number 2; l. VH of sequence number 6 and VL of sequence number 3; m. VH of sequence number 6 and VL of sequence number 140; n. VH of sequence number 6 and VL of sequence number 160; o. VH of sequence number 139 and VL of sequence number 1; p. VH of sequence number 139 and VL of sequence number 2; q. VH of sequence number 139 and VL of sequence number 3; r. VH of sequence number 139 and VL of sequence number 140; s. VH of sequence number 139 and VL of sequence number 160; t. VH of sequence number 159 and VL of sequence number 1; u. VH of sequence number 159 and VL of sequence number 2; v. VH of sequence number 159 and VL of sequence number 3; w. VH of sequence number 159 and VL of sequence number 140; x. VH of sequence number 159 and VL of sequence number 160; y. VH of sequence number 161 and VL of sequence number 1; z. VH of sequence number 159 and VL of sequence number 2; aa. VH of sequence number 161 and VL of sequence number 3; bb. VH of SEQ ID NO: 161 and VL of SEQ ID NO: 140; or cc. VH of sequence number 161 and VL of sequence number 160.

217. The antigen-binding domain that binds to the hK2 is scFV, according to claims 204 to 216. The CAR listed in any one of the items.

218. Claim 217, wherein the scFv includes a first linker (L1) between VL and VH. The CAR as described.

219. The CAR according to claim 218, wherein L1 comprises the amino acid sequence of SEQ ID NO:

7.

220. The aforementioned scFV is sequence numbers 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 、１８、１９、２０、２１、２２、２３、１３３、１３４、１３５、１３６、３０８、３ １６、３１８、３１９、３２０、３２１、３２２、３２３、３２４、３２５、４０４、４ The present invention, as described in claim 219, comprising the amino acid sequence 05, 406, 407, 408, or 409. The CAR.

221. The scFV has at least 80 amino acid sequences from the amino acid sequence of SEQ ID NO: 404 or SEQ ID NO:

405. % (for example, at least 90%, at least 95%, at least 98%, at least 99%) The CAR according to claim 219, comprising an amino acid sequence that is identical in %) or 100%.

222. The aforementioned scFv is sequence number 133, sequence number 134, sequence number 135, or sequence number 1 36 amino acid sequences and at least 80% (e.g., at least 90%, at least 95%) Contains amino acid sequences that are identical by at least 98%, at least 99%, or 100%. , the CAR according to claim 219.

223. The extracellular domain, which includes an antigen-binding domain that binds to hK2, is a signaling polyp A CAR according to any one of claims 217 to 222, further comprising a petit de.

224. The signal polypeptide comprises the amino acid sequence of SEQ ID NO: 24, as described in claim 223. Carrying a car.

225. The intracellular signaling domain is a TNF receptor superfamily member 9(C) D137) component, T cell surface glycoprotein CD3 zeta chain (CD3z) component, surface antigen component (CD27) components, surface antigen classification superfamily member components, and combinations thereof Any of claims 204 to 224, comprising a polypeptide component selected from the group consisting of combinations The CAR listed in any one of the items.

226. The CD137 component comprises the amino acid sequence of SEQ ID NO: 27, according to claims 204-225. The CAR listed in any one of the items.

227. The CD3z component comprises the amino acid sequence of SEQ ID NO: 28, according to claims 204 to 226. The CAR described in either item.

228. Claim 20, wherein the intracellular signaling domain comprises the amino acid sequence of Sequence ID No.

45. A CAR as described in any one of items 4 to 227.

229. Claim 20, wherein the CD8a-TM polypeptide comprises the amino acid sequence of SEQ ID NO: 26 A CAR as described in any one of items 4 to 228.

230. Claims 204-22, wherein the CD8a hinge region includes the amino acid sequence of Sequence ID No.

25. A CAR as described in any one of item 9.

231. The extracellular domain, which includes an antigen-binding domain that binds to hK2, is, SEQ ID NO: 29, ３０、３１、３２、３３、３４、３５、３６、３７、３８、３９、４０、４１、４２、４ ３、４４、１４９、１５０、１５１、１５２、４１０、４１１、４１２、４１３、４１４ 、４１５、４１６、４１７、４１８、４１９、４２０、４２１、４２２、４２３、４２４ or the CA according to any one of claims 223 to 230, comprising an amino acid sequence of 425. R.

232. The extracellular domain, which includes an antigen-binding domain that binds to hK2, is SEQ ID NO: 416 Or the amino acid sequence of SEQ ID NO: 417 and at least 80% (for example, at least 90%, less (At least 95%, at least 98%, at least 99%, or 100%) identical mesh A CAR according to any one of claims 223 to 230, comprising a no-acid sequence.

233. The extracellular domain, which includes the antigen-binding domain that binds to hK2, is SEQ ID NO: 149 , the amino acid sequence of SEQ ID NO: 150, SEQ ID NO: 151, or SEQ ID NO: 152 and at least 8 0% (for example, at least 90%, at least 95%, at least 98%, at least 9 Any of claims 223 to 230, comprising an amino acid sequence that is identical by 9% or 100%. The CAR listed.

234. Sequence numbers 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 、５８、５９、６０、６１、１５３、１５４、１５５、１５６、４２６、４２７、４２８ 、４２９、４３０、４３１、４３２、４３３、４３４、４３５、４３５、４３６、４３７ Claims 204 to 233, comprising the amino acid sequence 438, 439, 440, or 441 The CAR listed in any one of the items.

235. The amino acid sequence of SEQ ID NO: 432 or SEQ ID NO: 433 and at least 80% (for example, less At least 90%, at least 95%, at least 98%, at least 99%, or 100% ) The CAR according to any one of claims 204 to 233, comprising the same amino acid sequence 。

236. The amino acid combination of SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, or SEQ ID NO: 156 Columns and at least 80% (e.g., at least 90%, at least 95%, at least 98%) Claims 204 to 204 include amino acid sequences that are identical by %), at least 99%, or 100%. A CAR as described in any one of paragraphs 233.

237. Isolated lymphocytes expressing the CAR described in any one of claims 204 to 236.

238. The isolated lymphocyte according to claim 237, wherein the lymphocyte is a T lymphocyte.

239. The isolated lymphocytes according to claim 238 are natural killer (NK) cells. N-cell.

240. An isolated polynucleotide encoding the CAR according to any one of claims 204 to 236 Leotid.

241. A vector comprising the polynucleotide described in claim 240.

242. A dwelling containing the polynucleotide described in claim 240 or the vector described in claim 241. Principal cell.

243. Lymphocytes according to any one of claims 237 to 239 and a pharmaceutically acceptable excipient A pharmaceutical composition containing the following.

244. A method for treating a subject having hK2-expressing cancer, wherein the effective amount of treatment is as described in claims 237-23. Administer the lymphocytes described in any one of item 9 to a subject in need, thereby The lymphocytes mediate the killing of the hK2-expressing cancer in the subject, method.

245. The method according to claim 244, wherein the hK2-expressing cancer is prostate cancer or breast cancer.

246. The method according to claim 245, wherein the hK2-expressing cancer is prostate cancer.

247. A method for targeting and killing cancer cells, wherein the cancer cells are... (any of claims 244 to 246) By bringing the lymphocytes described in item 1 into contact with the cancer cells, the lymphocytes will be targeted and killed. A method that includes inducing injury.

248. The method according to claim 247, wherein the cancer cells are prostate cancer cells or breast cancer cells.

249. The method according to claim 248, wherein the cancer cells are prostate cancer cells.

250. A method for detecting the presence of cancer in a subject, a. A cell sample obtained from the subject as described in any one of claims 204 to 236. By bringing it into contact with the CAR, a CAR-cell complex is formed, b. To detect the CAR-cell complex, and here, the detection of the CAR-cell complex The result indicates the presence of the cancer in the subject. Methods that include...

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