Treatment of bone marrow disorders and acute leukemia targeting novel tumor-specific antigens

Abstract

The present disclosure relates to methods of diagnosing or treating bone marrow disorders and acute leukemia by using a tumor-specific antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109. Antigen binding proteins (ABPs), ABP-drug conjugates, and CARs targeting tumor specific antigens are also provided, as are methods of their use.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 327,757, filed on April 5, 2022, and U.S. Provisional Application No. 63 / 478,068, filed on December 30, 2022, which are incorporated herein by reference in their entireties.

[0002] 1. Sequence Listing

[0003] This application contains a sequence listing, which is submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on April 3, 2023 and is named 34619-55205_WO (005WO) and is 1,66,998 bytes in size. 2. Background technology

[0004] Acute myeloid leukemia (AML) is a blood cancer that originates in the bone marrow and accounts for approximately one-third of all childhood malignancies. AML causes the largest number of cancer-related deaths in children, with an overall survival rate of 55-70% at 5 years of follow-up. For more than 40 years, chemotherapy has been the standard AML treatment, and although it often causes the cancer to go into remission, it rarely completely eliminates the cancer cells. This often leads to disease relapse and ultimately patient death because second-line treatments are limited. After achieving a first remission, more than 50% of patients currently take aggressive post-remission treatments, such as high-dose chemotherapy and hematopoietic cell transplants. For many relapsed patients who are not healthy enough to tolerate such aggressive post-remission treatments, there are no available treatment options.

[0005] Therefore, a great deal of effort has been made to develop alternative primary and post-remission treatments for AML, but without significant success. AML cancer cells show changes in transcription factor occupancy and transcriptional regulation, and AML patients have different symptoms and prognoses associated with different subtypes of leukemia. It has been proposed that targeting specific subtypes of leukemia may allow for more effective personalized treatment. However, the subtype of leukemia in an individual patient can change over time or as a result of treatment, and an individual patient can have multiple subclones.

[0006] Immunotherapeutic approaches to treat AML have also been studied. Interleukin-3 receptor alpha chain (IL3RA, also known as CD123) is one of the first targeted antigens for the treatment of AML because it is overexpressed on the vast majority of AML cells compared to normal bone marrow. Monoclonal antibodies and recombinant immunotoxins targeting CD123 have shown promise in preclinical evaluations. CD33 is another target antigen because it is expressed on more than 80% of AML malignant cells; however, it is also expressed on normal bone marrow progenitor cell lines. Based on promising preclinical data, immunotherapies targeting these two surface molecules, including CAR-T cells, have been tested in early clinical trials in patients with relapsed and refractory AML. However, preliminary clinical results have been disappointing because these approaches have shown only short-term responses with no long-term benefit to the disease and have triggered severe side effects due to the poor specificity of the targets used, i.e., severe pancytopenia and severe bone marrow cell ablation due to the fact that hematopoietic stem and progenitor cells also express the target.

[0007] Therefore, there is a need to develop safe and effective alternative therapies for the treatment of AML. 1. Summary of the invention

[0008] The applicant used computer simulation analysis, followed by experimental analysis and validation, to identify six new antigens (i.e., CD63, CD151, CD72, CD84, CD69, and CD109) that are specific to AML cells and are barely or not expressed in the hematopoietic stem cell compartment. The six tumor-specific antigens (TSAs) were selected based in part on their stable, specific, and high-level expression in AML cells as determined by flow cytometry using AML cell lines (SHI-1, HL-60, KASUMI-1, MOLM-13, MV4-1, and AML).

[0009] Based on this discovery, the applicant provides a method for diagnosing and treating bone marrow disorders and acute leukemia (e.g., AML) by targeting TSAs selected from CD63, CD151, CD72, CD84, CD69, and CD109. Antigen binding proteins (ABPs), ABP-drug conjugates, and chimeric antigen receptors (CARs) that can be used in therapeutic methods are also provided herein.

[0010] Accordingly, the present disclosure provides a method for diagnosing myeloid disorders (MD) and acute leukemia (AL) in a subject, the method comprising the following steps: detecting the presence or level of a tumor-specific antigen in a biological sample of the subject, wherein the tumor-specific antigen is selected from CD63, CD151, CD72, CD84, CD69 and CD109.

[0011] In some embodiments, the biological sample is a blood sample or a bone marrow sample. In some embodiments, the biological sample comprises blasts. In some embodiments, the blasts are selected from bone marrow blasts, lymphoid blasts, or a combination of bone marrow and lymphoid blasts.

[0012] In some embodiments, the detection step includes contacting the biological sample with an antibody, wherein the antibody specifically binds to a tumor-specific antigen. In some embodiments, the detection step includes flow cytometry, immunocytochemistry, immunohistochemistry, fluorescence or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the antibody is labeled. In some embodiments, the antibody is labeled with a fluorophore or an enzyme. In some embodiments, the target binding protein is labeled. In some embodiments, the target binding protein is labeled with a fluorophore or an enzyme. In some embodiments, the detection step includes measuring the mRNA level of the tumor-specific antigen in the biological sample.

[0013] In some embodiments, the detecting step comprises measuring the mRNA level of the tumor-specific antigen in the biological sample. In some embodiments, the mRNA level is measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR) or by next generation sequencing.

[0014] In some embodiments, myeloid disorders (MD) and acute leukemia (AL) occur in childhood or adult age. In some embodiments, the method further comprises a step of determining the presence or absence of cancer cells in the subject based on detecting the presence or level of a tumor-specific antigen in a blood and / or bone marrow sample from the subject. In some embodiments, the method further comprises a step of determining the presence or absence of cancer cells in the subject based on detecting the presence or level of a tumor-specific antigen in a biological sample from the subject.

[0015] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a therapeutic agent that specifically binds to a tumor-specific antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109.

[0016] In some embodiments, wherein the therapeutic agent is an antigen binding protein (ABP), an ABP-drug conjugate, an immune response cell expressing a chimeric antigen receptor (CAR), or a bispecific T cell engager (BiTE), wherein the ABP, ABP-drug conjugate, CAR, or BiTE specifically binds to a tumor-specific antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109. In some embodiments, wherein the therapeutic agent is an ABP described in the present disclosure, an ABP-drug conjugate described in the present disclosure, an immune response cell expressing a chimeric antigen receptor (CAR) described in the present disclosure, or a bispecific T cell engager (BiTE) described in the present disclosure.

[0017] In another aspect, the present disclosure provides a method for treating a subject with a myeloid disorder (MD) or acute leukemia (AL), the method comprising: a) detecting the presence or level of a tumor-specific antigen in a biological sample of the subject, wherein the tumor-specific antigen is selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109; b) administering to the subject a therapeutically effective amount of an antigen binding protein (ABP), an ABP-drug conjugate, or an immune response cell expressing a chimeric antigen receptor (CAR), wherein the ABP, ABP-drug conjugate, or CAR specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0018] In some embodiments, the biological sample is a blood sample, a bone marrow sample. In some embodiments, the biological sample comprises myeloid disorder (MD) and acute leukemia (AL) blasts. In some embodiments, the blasts are selected from myeloid blasts, lymphoid blasts, or a combination of myeloid and lymphoid blasts.

[0019] In some embodiments, the presence or level of a tumor-specific antigen is detected by contacting a biological sample with an antibody, wherein the antibody specifically binds to the tumor-specific antigen. In some embodiments, the presence or level of a tumor-specific antigen is detected by flow cytometry, immunocytochemistry, immunohistochemistry, fluorescence, or enzyme-linked immunosorbent assay (ELISA).

[0020] In some embodiments, the presence or level of a tumor-specific antigen is detected by measuring the mRNA level of the tumor-specific antigen in a biological sample. In some embodiments, the mRNA level is measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR) or next generation sequencing.

[0021] In one aspect, the present disclosure provides an antigen binding protein (ABP) that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0022] In some embodiments, the ABP specifically binds human CD84.

[0023] In some embodiments, the amino acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:97, SEQ ID NO:98 or SEQ ID NO:90.

[0024] In some embodiments, the ABP comprises:

[0025] a. V having the sequence of SEQ ID NO: 51 L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 61 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3;

[0026] b. V having the sequence of SEQ ID NO: 47 L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3; or

[0027] c. V having the sequence of SEQ ID NO: 45 L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 63 HCDR1, V having the sequence of SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0028] In some embodiments, the ABP comprises:

[0029] d. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88;

[0030] e. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:81, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:88; or

[0031] f. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74 and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:83.

[0032] In some embodiments, the ABP comprises

[0033] a. V having the sequence of SEQ ID NO: 131 L CDR1, V having the sequence of SEQ ID NO: 132 L CDR2, V having the sequence of SEQ ID NO: 133 L CDR3, V having the sequence of SEQ ID NO: 251 H CDR1, V having the sequence of SEQ ID NO: 252 H CDR2 and V having the sequence of SEQ ID NO: 253 H CDR3;

[0034] b. V having the sequence of SEQ ID NO: 134 L CDR1, V having the sequence of SEQ ID NO: 135 L CDR2, V having the sequence of SEQ ID NO: 136 L CDR3, V having the sequence of SEQ ID NO: 254 H CDR1, V having the sequence of SEQ ID NO: 255 H CDR2 and V having the sequence of SEQ ID NO: 256 H CDR3;

[0035] c. V having the sequence of SEQ ID NO: 110 L CDR1, V having the sequence of SEQ ID NO: 111 L CDR2, V having the sequence of SEQ ID NO: 112 L CDR3, V having the sequence of SEQ ID NO: 230 H CDR1, V having the sequence of SEQ ID NO: 231 H CDR2 and V having the sequence of SEQ ID NO: 232 H CDR3;

[0036] d. V having the sequence of SEQ ID NO: 161 L CDR1, V having the sequence of SEQ ID NO: 162 L CDR2, V having the sequence of SEQ ID NO: 163 L CDR3, V having the sequence of SEQ ID NO: 281 H CDR1, V having the sequence of SEQ ID NO: 282 H CDR2 and V having the sequence of SEQ ID NO: 283 H CDR3;

[0037] e. V having the sequence of SEQ ID NO: 164 L CDR1, V having the sequence of SEQ ID NO: 165 L CDR2, V having the sequence of SEQ ID NO: 166 L CDR3, V having the sequence of SEQ ID NO: 284 H CDR1, V having the sequence of SEQ ID NO: 285 HCDR2 and V having the sequence of SEQ ID NO: 286 H CDR3;

[0038] f. V having the sequence of SEQ ID NO: 140 L CDR1, V having the sequence of SEQ ID NO: 141 L CDR2, V having the sequence of SEQ ID NO: 142 L CDR3, V having the sequence of SEQ ID NO: 260 H CDR1, V having the sequence of SEQ ID NO: 261 H CDR2 and V having the sequence of SEQ ID NO: 262 H CDR3;

[0039] g. V having the sequence of SEQ ID NO: 191 L CDR1, V having the sequence of SEQ ID NO: 192 L CDR2, V having the sequence of SEQ ID NO: 193 L CDR3, V having the sequence of SEQ ID NO: 311 H CDR1, V having the sequence of SEQ ID NO: 312 H CDR2 and V having the sequence of SEQ ID NO: 313 H CDR3;

[0040] h. V having the sequence of SEQ ID NO: 194 L CDR1, V having the sequence of SEQ ID NO: 195 L CDR2, V having the sequence of SEQ ID NO: 196 L CDR3, V having the sequence of SEQ ID NO: 314 H CDR1, V having the sequence of SEQ ID NO: 315 H CDR2 and V having the sequence of SEQ ID NO: 316 H CDR3;

[0041] i. V having the sequence of SEQ ID NO: 170 L CDR1, V having the sequence of SEQ ID NO: 171 L CDR2, V having the sequence of SEQ ID NO: 172 L CDR3, V having the sequence of SEQ ID NO: 290H CDR1, V having the sequence of SEQ ID NO: 291 H CDR2 and V having the sequence of SEQ ID NO: 292 H CDR3;

[0042] j. V having the sequence of SEQ ID NO: 221 L CDR1, V having the sequence of SEQ ID NO: 222 L CDR2, V having the sequence of SEQ ID NO: 223 L CDR3, V having the sequence of SEQ ID NO: 341 H CDR1, V having the sequence of SEQ ID NO: 342 H CDR2 and V having the sequence of SEQ ID NO: 343 H CDR3; or

[0043] V having the sequence of SEQ ID NO: 224 L CDR1, V having the sequence of SEQ ID NO: 225 L CDR2, V having the sequence of SEQ ID NO: 226 L CDR3, V having the sequence of SEQ ID NO: 344 H CDR1, V having the sequence of SEQ ID NO: 345 H CDR2 and V having the sequence of SEQ ID NO: 346 H CDR3; or

[0044] k. V having the sequence of SEQ ID NO: 200 L CDR1, V having the sequence of SEQ ID NO: 201 L CDR2, V having the sequence of SEQ ID NO: 202 L CDR3, V having the sequence of SEQ ID NO: 320 H CDR1, V having the sequence of SEQ ID NO: 321 H CDR2 and V having the sequence of SEQ ID NO: 322 H CDR3.

[0045] In some embodiments, the ABP specifically binds human CD69.

[0046] In some embodiments, the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 89-96.

[0047] In some embodiments, the ABP comprises:

[0048] a. V having the sequence of SEQ ID NO: 47 L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 57 L CDR3, V having the sequence of SEQ ID NO: 64 H CDR1, V having the sequence of SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3;

[0049] b. V having the sequence of SEQ ID NO: 50 L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 60 L CDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO: 69 H CDR2 and V having the sequence of SEQ ID NO: 73 H CDR3;

[0050] c. V having the sequence of SEQ ID NO: 45 L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO: 65 H CDR2 and V having the sequence of SEQ ID NO: 70 H CDR3;

[0051] d. V having the sequence of SEQ ID NO: 46 L CDR1, V having the sequence of SEQ ID NO: 53 LCDR2, V having the sequence of SEQ ID NO: 56 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3;

[0052] e. V having the sequence of SEQ ID NO: 48 L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 58 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3;

[0053] f. V having the sequence of SEQ ID NO: 49 L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3;

[0054] g. V having the sequence of SEQ ID NO: 49 L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3; or

[0055] h. V having the sequence of SEQ ID NO: 45 LCDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0056] In some embodiments, the ABP comprises:

[0057] a. V having the sequence of SEQ ID NO: 107 L CDR1, V having the sequence of SEQ ID NO: 108 L CDR2, V having the sequence of SEQ ID NO: 109 L CDR3, V having the sequence of SEQ ID NO: 227 H CDR1, V having the sequence of SEQ ID NO: 228 H CDR2 and V having the sequence of SEQ ID NO: 229 H CDR3;

[0058] b. V having the sequence of SEQ ID NO: 110 L CDR1, V having the sequence of SEQ ID NO: 111 L CDR2, V having the sequence of SEQ ID NO: 112 L CDR3, V having the sequence of SEQ ID NO: 230 H CDR1, V having the sequence of SEQ ID NO: 231 H CDR2 and V having the sequence of SEQ ID NO: 232 H CDR3;

[0059] c. V having the sequence of SEQ ID NO: 113 L CDR1, V having the sequence of SEQ ID NO: 114 L CDR2, V having the sequence of SEQ ID NO: 115 L CDR3, V having the sequence of SEQ ID NO: 233 H CDR1, V having the sequence of SEQ ID NO: 234 HCDR2 and V having the sequence of SEQ ID NO: 235 H CDR3;

[0060] d. V having the sequence of SEQ ID NO: 116 L CDR1, V having the sequence of SEQ ID NO: 117 L CDR2, V having the sequence of SEQ ID NO: 118 L CDR3, V having the sequence of SEQ ID NO: 236 H CDR1, V having the sequence of SEQ ID NO: 237 H CDR2 and V having the sequence of SEQ ID NO: 238 H CDR3;

[0061] e. V having the sequence of SEQ ID NO: 119 L CDR1, V having the sequence of SEQ ID NO: 120 L CDR2, V having the sequence of SEQ ID NO: 121 L CDR3, V having the sequence of SEQ ID NO: 239 H CDR1, V having the sequence of SEQ ID NO: 240 H CDR2 and V having the sequence of SEQ ID NO: 241 H CDR3;

[0062] f. V having the sequence of SEQ ID NO: 122 L CDR1, V having the sequence of SEQ ID NO: 123 L CDR2, V having the sequence of SEQ ID NO: 124 L CDR3, V having the sequence of SEQ ID NO: 242 H CDR1, V having the sequence of SEQ ID NO: 243 H CDR2 and V having the sequence of SEQ ID NO: 244 H CDR3;

[0063] g. V having the sequence of SEQ ID NO: 125 L CDR1, V having the sequence of SEQ ID NO: 126 L CDR2, V having the sequence of SEQ ID NO: 127 L CDR3, V having the sequence of SEQ ID NO: 245H CDR1, V having the sequence of SEQ ID NO: 246 H CDR2 and V having the sequence of SEQ ID NO: 247 H CDR3;

[0064] h. V having the sequence of SEQ ID NO: 128 L CDR1, V having the sequence of SEQ ID NO: 129 L CDR2, V having the sequence of SEQ ID NO: 130 L CDR3, V having the sequence of SEQ ID NO: 248 H CDR1, V having the sequence of SEQ ID NO: 249 H CDR2 and V having the sequence of SEQ ID NO: 250 H CDR3;

[0065] i. V having the sequence of SEQ ID NO: 137 L CDR1, V having the sequence of SEQ ID NO: 138 L CDR2, V having the sequence of SEQ ID NO: 139 L CDR3, V having the sequence of SEQ ID NO: 257 H CDR1, V having the sequence of SEQ ID NO: 258 H CDR2 and V having the sequence of SEQ ID NO: 259 H CDR3;

[0066] j. V having the sequence of SEQ ID NO: 140 L CDR1, V having the sequence of SEQ ID NO: 141 L CDR2, V having the sequence of SEQ ID NO: 142 L CDR3, V having the sequence of SEQ ID NO: 260 H CDR1, V having the sequence of SEQ ID NO: 261 H CDR2 and V having the sequence of SEQ ID NO: 262 H CDR3;

[0067] k. V having the sequence of SEQ ID NO: 143 L CDR1, V having the sequence of SEQ ID NO: 144 LCDR2, V having the sequence of SEQ ID NO: 145 L CDR3, V having the sequence of SEQ ID NO: 263 H CDR1, V having the sequence of SEQ ID NO: 264 H CDR2 and V having the sequence of SEQ ID NO: 265 H CDR3;

[0068] 1. V having the sequence of SEQ ID NO: 146 L CDR1, V having the sequence of SEQ ID NO: 147 L CDR2, V having the sequence of SEQ ID NO: 148 L CDR3, V having the sequence of SEQ ID NO: 266 H CDR1, V having the sequence of SEQ ID NO: 267 H CDR2 and V having the sequence of SEQ ID NO: 268 H CDR3;

[0069] m. V having the sequence of SEQ ID NO: 149 L CDR1, V having the sequence of SEQ ID NO: 150 L CDR2, V having the sequence of SEQ ID NO: 151 L CDR3, V having the sequence of SEQ ID NO: 269 H CDR1, V having the sequence of SEQ ID NO: 270 H CDR2 and V having the sequence of SEQ ID NO: 271 H CDR3;

[0070] n. V having the sequence of SEQ ID NO: 152 L CDR1, V having the sequence of SEQ ID NO: 153 L CDR2, V having the sequence of SEQ ID NO: 154 L CDR3, V having the sequence of SEQ ID NO: 272 H CDR1, V having the sequence of SEQ ID NO: 273 H CDR2 and V having the sequence of SEQ ID NO: 274 H CDR3;

[0071] o. V having the sequence of SEQ ID NO: 155L CDR1, V having the sequence of SEQ ID NO: 156 L CDR2, V having the sequence of SEQ ID NO: 157 L CDR3, V having the sequence of SEQ ID NO: 275 H CDR1, V having the sequence of SEQ ID NO: 276 H CDR2 and V having the sequence of SEQ ID NO: 277 H CDR3;

[0072] p. V having the sequence of SEQ ID NO: 158 L CDR1, V having the sequence of SEQ ID NO: 159 L CDR2, V having the sequence of SEQ ID NO: 160 L CDR3, V having the sequence of SEQ ID NO: 278 H CDR1, V having the sequence of SEQ ID NO: 279 H CDR2 and V having the sequence of SEQ ID NO: 280 H CDR3;

[0073] q. V having the sequence of SEQ ID NO: 167 L CDR1, V having the sequence of SEQ ID NO: 168 L CDR2, V having the sequence of SEQ ID NO: 169 L CDR3, V having the sequence of SEQ ID NO: 287 H CDR1, V having the sequence of SEQ ID NO: 288 H CDR2 and V having the sequence of SEQ ID NO: 289 H CDR3;

[0074] r. V having the sequence of SEQ ID NO: 170 L CDR1, V having the sequence of SEQ ID NO: 171 L CDR2, V having the sequence of SEQ ID NO: 172 L CDR3, V having the sequence of SEQ ID NO: 290 H CDR1, V having the sequence of SEQ ID NO: 291 H CDR2 and V having the sequence of SEQ ID NO: 292 H CDR3;

[0075] s. V having the sequence of SEQ ID NO: 173 L CDR1, V having the sequence of SEQ ID NO: 174 L CDR2, V having the sequence of SEQ ID NO: 175 L CDR3, V having the sequence of SEQ ID NO: 293 H CDR1, V having the sequence of SEQ ID NO: 294 H CDR2 and V having the sequence of SEQ ID NO: 295 H CDR3;

[0076] t. V having the sequence of SEQ ID NO: 176 L CDR1, V having the sequence of SEQ ID NO: 177 L CDR2, V having the sequence of SEQ ID NO: 178 L CDR3, V having the sequence of SEQ ID NO: 296 H CDR1, V having the sequence of SEQ ID NO: 297 H CDR2 and V having the sequence of SEQ ID NO: 298 H CDR3;

[0077] u. V having the sequence of SEQ ID NO: 179 L CDR1, V having the sequence of SEQ ID NO: 180 L CDR2, V having the sequence of SEQ ID NO: 181 L CDR3, V having the sequence of SEQ ID NO: 299 H CDR1, V having the sequence of SEQ ID NO: 300 H CDR2 and V having the sequence of SEQ ID NO: 301 H CDR3;

[0078] v. V having the sequence of SEQ ID NO: 182 L CDR1, V having the sequence of SEQ ID NO: 183 L CDR2, V having the sequence of SEQ ID NO: 184 L CDR3, V having the sequence of SEQ ID NO: 302 H CDR1, V having the sequence of SEQ ID NO: 303 HCDR2 and V having the sequence of SEQ ID NO: 304 H CDR3;

[0079] w. V having the sequence of SEQ ID NO: 185 L CDR1, V having the sequence of SEQ ID NO: 186 L CDR2, V having the sequence of SEQ ID NO: 187 L CDR3, V having the sequence of SEQ ID NO: 305 H CDR1, V having the sequence of SEQ ID NO: 306 H CDR2 and V having the sequence of SEQ ID NO: 307 H CDR3;

[0080] x. V having the sequence of SEQ ID NO: 188 L CDR1, V having the sequence of SEQ ID NO: 189 L CDR2, V having the sequence of SEQ ID NO: 190 L CDR3, V having the sequence of SEQ ID NO: 308 H CDR1, V having the sequence of SEQ ID NO: 309 H CDR2 and V having the sequence of SEQ ID NO: 310 H CDR3;

[0081] y. V having the sequence of SEQ ID NO: 197 L CDR1, V having the sequence of SEQ ID NO: 198 L CDR2, V having the sequence of SEQ ID NO: 199 L CDR3, V having the sequence of SEQ ID NO: 317 H CDR1, V having the sequence of SEQ ID NO: 318 H CDR2 and V having the sequence of SEQ ID NO: 319 H CDR3;

[0082] z. V having the sequence of SEQ ID NO: 200 L CDR1, V having the sequence of SEQ ID NO: 201 L CDR2, V having the sequence of SEQ ID NO: 202 L CDR3, V having the sequence of SEQ ID NO: 320H CDR1, V having the sequence of SEQ ID NO: 321 H CDR2 and V having the sequence of SEQ ID NO: 322 H CDR3;

[0083] aa. V having a sequence of SEQ ID NO: 203 L CDR1, V having the sequence of SEQ ID NO: 204 L CDR2, V having the sequence of SEQ ID NO: 205 L CDR3, V having the sequence of SEQ ID NO: 323 H CDR1, V having the sequence of SEQ ID NO: 324 H CDR2 and V having the sequence of SEQ ID NO: 325 H CDR3;

[0084] bb. V having the sequence of SEQ ID NO: 206 L CDR1, V having the sequence of SEQ ID NO: 207 L CDR2, V having the sequence of SEQ ID NO: 208 L CDR3, V having the sequence of SEQ ID NO: 326 H CDR1, V having the sequence of SEQ ID NO: 327 H CDR2 and V having the sequence of SEQ ID NO: 328 H CDR3;

[0085] cc. V having the sequence of SEQ ID NO: 209 L CDR1, V having the sequence of SEQ ID NO: 210 L CDR2, V having the sequence of SEQ ID NO: 211 L CDR3, V having the sequence of SEQ ID NO: 329 H CDR1, V having the sequence of SEQ ID NO: 330 H CDR2 and V having the sequence of SEQ ID NO: 331 H CDR3;

[0086] dd. V having the sequence of SEQ ID NO: 212 L CDR1, V having the sequence of SEQ ID NO: 213 LCDR2, V having the sequence of SEQ ID NO: 214 L CDR3, V having the sequence of SEQ ID NO: 332 H CDR1, V having the sequence of SEQ ID NO: 333 H CDR2 and V having the sequence of SEQ ID NO: 334 H CDR3;

[0087] ee. V having the sequence of SEQ ID NO: 215 L CDR1, V having the sequence of SEQ ID NO: 216 L CDR2, V having the sequence of SEQ ID NO: 217 L CDR3, V having the sequence of SEQ ID NO: 335 H CDR1, V having the sequence of SEQ ID NO: 336 H CDR2 and V having the sequence of SEQ ID NO: 337 H CDR3; or

[0088] ff. V having the sequence of SEQ ID NO: 218 L CDR1, V having the sequence of SEQ ID NO: 219 L CDR2, V having the sequence of SEQ ID NO: 220 L CDR3, V having the sequence of SEQ ID NO: 338 H CDR1, V having the sequence of SEQ ID NO: 339 H CDR2 and V having the sequence of SEQ ID NO: 340 H CDR3.

[0089] In some embodiments, the ABP comprises:

[0090] a. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 76, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85;

[0091] b. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 87;

[0092] c. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83;

[0093] d. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 82;

[0094] e. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 75, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84;

[0095] f. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86;

[0096] g. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84;

[0097] h. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 78, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84; or

[0098] i. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:83.

[0099] In some embodiments, the ABP specifically binds human CD69 and CD84.

[0100] In some embodiments, the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:36.

[0101] In some embodiments, the ABP comprises:

[0102] V having the sequence of SEQ ID NO:45 L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0103] In some embodiments, the ABP comprises a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:83.

[0104] In some embodiments, the ABP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-98.

[0105] In some embodiments, the ABP comprises a Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V domain antibody.

[0106] In some embodiments, the ABP is a Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V domain antibody.

[0107] In some embodiments, the ABP is a monoclonal antibody.

[0108] In some embodiments, the ABP is selected from IgG, IgM, IgA, IgD, and IgE antibodies.

[0109] In some embodiments, the ABP comprises a heavy chain constant region of IgG class and a subclass selected from IgGl, IgG2, IgG3, and IgG4.

[0110] In some embodiments, the ABP is conjugated to a drug.

[0111] In some embodiments, the ABP is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), wherein the ABP specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, and wherein the ABP comprises a human Fc.

[0112] In some embodiments, the ABP is human, humanized, or chimeric.

[0113] In some embodiments, the ABP is monoclonal.

[0114] In some embodiments, the ABP is bispecific or multispecific.

[0115] In some embodiments, the ABP comprises the heavy chain constant region of IgG.

[0116] In some embodiments, the ABP is afucosylated.

[0117] In some embodiments, the ABP has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D Binds to the target protein.

[0118] In one aspect, the present disclosure provides an isolated polynucleotide or a set of isolated polynucleotides encoding the ABP described in the present disclosure. In another aspect, the present disclosure provides a vector or a set of vectors comprising the isolated polynucleotide described in the present disclosure. In another aspect, the present disclosure provides a host cell comprising the isolated polynucleotide or vector described in the present disclosure.

[0119] In one aspect, the present disclosure provides a method of producing an isolated antigen binding protein (ABP), comprising expressing the ABP in a host cell described in the present disclosure and isolating the ABP.

[0120] In one aspect, the present disclosure provides an antigen binding protein (ABP) capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), wherein the ABP specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, and wherein the ABP comprises a human Fc.

[0121] In some embodiments, the ABP is human, humanized or chimeric. In some embodiments, the ABP is monoclonal. In some embodiments, the ABP is bispecific or multispecific. In some embodiments, the ABP comprises the heavy chain constant region of IgG. In some embodiments, the ABP comprises the heavy chain constant region of IgG1. In some embodiments, the ABP is afucosylated.

[0122] In some embodiments, the ABP has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D Binds to the target protein.

[0123] In another aspect, the disclosure provides a pharmaceutical composition comprising the ABP provided herein and a pharmaceutically acceptable excipient.

[0124] In yet another aspect, the present disclosure provides a method of treating a subject with a myeloid disorder (MD) or acute leukemia (AL), the method comprising administering a therapeutically effective amount of an ABP or pharmaceutical composition provided herein. In some embodiments, the myeloid disorder (MD) and acute leukemia (AL) are pediatric or adult onset.

[0125] In some embodiments, the ABP or pharmaceutical composition is administered in combination with an additional agent. In some embodiments, the additional agent is a chemotherapeutic agent or a biological agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacitidine, decitabine, and CPX-351. In some embodiments, the additional agent is a Hedgehog signaling pathway inhibitor. In some embodiments, the Hedgehog signaling pathway inhibitor is a Sonic Hedgehog signaling pathway inhibitor. In some embodiments, the Sonic Hedgehog signaling pathway inhibitor is selected from Vismodegib, Sonidegib, and arsenic trioxide (ATO). In some embodiments, the Hedgehog signaling pathway inhibitor is Daurismo TM In some embodiments, the additional agent is a FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from midostaurin. Gilteritinib In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib. Ensidipine In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg TM) or tariline-vadatuximab (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor. In some embodiments, the additional agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0126] In some embodiments, the ABP or pharmaceutical composition is not administered in combination with an immunotherapeutic agent. In some embodiments, the ABP or pharmaceutical composition is not administered with a combination therapy. In some embodiments, the ABP or pharmaceutical composition is not administered with immunotherapy. In some embodiments, the method includes administering a therapeutically effective amount of an immune response cell comprising a CAR, and the administration step is followed by no autologous / allogeneic hematopoietic stem cell therapy for hematopoiesis rescue, or is not combined with autologous / allogeneic hematopoietic stem cell therapy for hematopoiesis rescue.

[0127] In yet another aspect, the present disclosure provides an antigen binding protein (ABP)-drug conjugate comprising: an antigen binding protein (ABP), a cytotoxic agent linked to the ABP, and optionally a linker linking the cytotoxic agent to the ABP, wherein the ABP specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0128] In some embodiments, the ABP is human, humanized or chimeric. In some embodiments, the ABP is monoclonal. In some embodiments, the ABP is bispecific or multispecific. In some embodiments, the ABP comprises Fab, Fab ", F (ab") 2, Fv, scFv, (scFv) 2, single-chain antibody molecules, dual variable domain antibodies, single variable domain antibodies, linear antibodies, V domain antibodies, or bispecific tandem bivalent scFv or bispecific T cell adapter (BiTE). In some embodiments, the ABP comprises Fc, optionally human Fc. In some embodiments, the ABP comprises a class of heavy chain constant regions selected from IgG, IgA, IgD, IgE and IgM. In some embodiments, the ABP comprises IgG class and a heavy chain constant region of a subclass selected from IgG1, IgG2, IgG3 and IgG4. In some embodiments, the ABP comprises a heavy chain constant region of IgG1.

[0129] In certain embodiments, the ABP is a BiTE. In specific embodiments, the BiTE comprises an antigen binding domain and a T cell activation domain. In certain embodiments, the antigen binding domain specifically binds to a target protein / antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109.

[0130] In some embodiments, the antigen binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109).

[0131] In some embodiments, the antigen binding domain binds to an epitope on a CD63, CD151, CD72, CD84, CD69, or CD109 target antigen. In some embodiments, the antigen binding domain comprises the CDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. In some embodiments, the antigen binding domain comprises the V H and V L In some embodiments, the antigen binding domain comprises a CD63, CD151, CD72, CD84, CD69 or CD109 single chain variable fragment (scFv).

[0132] In some embodiments, the T cell activation domain includes the intracellular domain of CD3ζ. In certain embodiments, the T cell activation domain binds CD3.

[0133] In some embodiments, the ABP has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D Binds to the target protein.

[0134] In some embodiments, the cytotoxic agent comprises an anti-angiogenic agent, a pro-apoptotic agent, an anti-mitotic agent, an anti-kinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioisotope. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker.

[0135] One aspect of the present disclosure provides a pharmaceutical composition comprising the ABP-drug conjugate described in the present disclosure and a pharmaceutically acceptable excipient.

[0136] In one aspect, the present disclosure provides a method of treating a subject suffering from a myeloid disorder (MD) or acute leukemia (AL), the method comprising: administering a therapeutically effective amount of an ABP-drug conjugate or pharmaceutical composition described in the present disclosure.

[0137] In some embodiments, myeloid disorders (MD) and acute leukemia (AL) are pediatric or adult onset. In some embodiments, the ABP-drug conjugate or pharmaceutical composition is administered in combination with an additional agent. In some embodiments, the additional agent is a chemotherapeutic agent or a biologic agent. In some embodiments, the chemotherapeutic agent is selected from cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacitidine, decitabine, and CPX-351. In some embodiments, the additional agent is a Hedgehog signaling pathway inhibitor. In some embodiments, the Hedgehog signaling pathway inhibitor is a Sonic Hedgehog signaling pathway inhibitor. In some embodiments, the Sonic Hedgehog signaling pathway inhibitor is selected from Vismodegib, Sonidegib, and arsenic trioxide (ATO). In some embodiments, the Hedgehog signaling pathway inhibitor is Daurismo TM In some embodiments, the additional agent is a FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from midostaurin. Gilteritinib In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib. Ensidipine In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg TM) or tariline-vadatuximab (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor. In some embodiments, the additional agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0138] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, a transmembrane domain, a signaling domain, and optionally a co-stimulatory domain, wherein the extracellular antigen binding domain specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0139] In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to the target protein.

[0140] In some embodiments, the extracellular antigen binding domain comprises an ABP described in the present disclosure.

[0141] In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ.

[0142] In some embodiments, the CAR further comprises a costimulatory domain, wherein the costimulatory domain is a CD28 costimulatory domain, a 4-1BB costimulatory domain, a CD27 costimulatory domain, an OX40 costimulatory domain, or an ICOS costimulatory domain.

[0143] In some embodiments, the costimulatory domain is a 4-1BB costimulatory domain. In some embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity with the amino acid sequence of SEQ ID NO: 105.

[0144] In some embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0145] In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:100.

[0146] In some embodiments, the CAR further comprises a hinge region.

[0147] In some embodiments, the hinge region is a hinge region derived from a CD28 polypeptide.

[0148] In some embodiments, the hinge region comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:99.

[0149] In some embodiments, the signaling domain is a CD3 zeta signaling domain.

[0150] In some embodiments, the CD3 zeta signaling domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO: 101.

[0151] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 35-44.

[0152] In some embodiments, the extracellular antigen binding domain specifically binds human CD84.

[0153] In some embodiments, the extracellular antigen binding domain specifically binds human CD69.

[0154] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid selected from SEQ ID NOs: 35-42.

[0155] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acids of SEQ ID NO:43, 44, or 36.

[0156] In one aspect, the present disclosure provides a polynucleotide encoding a CAR described in the present disclosure.

[0157] In some embodiments, the CAR comprises a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% sequence identity to the nucleotide sequence of SEQ ID NOs: 24-34.

[0158] In one aspect, the present disclosure provides a vector comprising the CAR polynucleotide of the present disclosure.

[0159] In one aspect, the present disclosure provides an immune response cell that expresses the CAR described in the present disclosure.

[0160] In one aspect, the present disclosure provides an immune response cell comprising the CAR polynucleotide described in the present disclosure or the vector described in the present disclosure.

[0161] In some embodiments, the immune response cell is an αβ T cell, a γδ T cell, or a natural killer (NK) cell.

[0162] In some embodiments, the αβ T cells are CD4 + T cells or CD8 + T cells.

[0163] In one aspect, the present disclosure provides a method for preparing the immune response cell described in the present disclosure, the method comprising transfecting or transducing the CAR polynucleotide or vector described in the present disclosure into an immune cell.

[0164] In some embodiments, the method comprises expanding the immune cells for at least 48 hours.

[0165] In some embodiments, the immune cells are transduced at a multiplicity of infection of 1 to 100.

[0166] In some embodiments, the method further comprises, after transduction, washing the vector, and amplifying the transduced immune cells for at least 2 days. In some embodiments, the method further comprises, after transduction, washing the vector, and amplifying the transduced immune cells for a period of 2-30 days.

[0167] In one aspect, the disclosure provides a method of treating a subject, the method comprising administering a therapeutically effective amount of an ABP described in the disclosure, an ABP-drug conjugate described in the disclosure, a pharmaceutical composition described in the disclosure, or an immune response cell described in the disclosure.

[0168] In some embodiments, the subject has a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, the acute leukemia is acute lymphoblastic leukemia (ALL).

[0169] In some embodiments, myeloid disorders (MD) and acute leukemias (AL) are pediatric or adult-onset.

[0170] In some embodiments, the extracellular antigen binding domain comprises a single-chain variable fragment (scFv) of an antibody that specifically binds to a target protein. In some embodiments, the signaling domain comprises an intracellular domain of CD3ζ. In some embodiments, CAR also comprises a costimulatory domain, wherein the costimulatory domain is a CD28 costimulatory domain, a 4-1BB costimulatory domain, a CD27 costimulatory domain, an OX40 costimulatory domain, or an ICOS costimulatory domain.

[0171] In one aspect, the present disclosure provides a polynucleotide encoding CAR. In another aspect, the present disclosure provides a vector comprising polynucleotides. In another aspect, the present disclosure provides an immune response cell expressing the CAR described in the present disclosure and comprising polynucleotides.

[0172] In some embodiments, the immune response cell is an αβT cell, a γδT cell, or a natural killer (NK) cell. In some embodiments, the αβT cell is a CD4 + T cells, CD3 + T cells or CD8 + T cells.

[0173] In one aspect, the present disclosure provides a method for preparing immune response cells, the method comprising transfecting or transducing a polynucleotide or a vector into the immune response cells.

[0174] In another aspect, the present disclosure provides a method of treating a subject with a myeloid disorder (MD) or acute leukemia (AL), the method comprising: administering a therapeutically effective amount of immune response cells. In some embodiments, the myeloid disorder (MD) and acute leukemia (AL) are pediatric or adult-onset.

[0175] In some embodiments, the immune response cells are administered in combination with additional agents. In some embodiments, the additional agents are administered before or after administering a therapeutically effective amount of immune response cells. In some embodiments, the additional agents are administered simultaneously with administering a therapeutically effective amount of immune response cells. In some embodiments, the additional agents are administered before administering a therapeutically effective amount of immune response cells. In some embodiments, the additional agents are administered after administering a therapeutically effective amount of immune response cells.

[0176] In some embodiments, the additional agent is a chemotherapeutic agent or a biologic agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacitidine, decitabine, and CPX-351 In some embodiments, the additional agent is a Hedgehog signaling pathway inhibitor. In some embodiments, the Hedgehog signaling pathway inhibitor is a Sonic Hedgehog signaling pathway inhibitor. In some embodiments, the Sonic Hedgehog signaling pathway inhibitor is selected from Vismodegib, Sonidegib, and arsenic trioxide (ATO). In some embodiments, the Hedgehog signaling pathway inhibitor is Daurismo TM In some embodiments, the additional agent is a FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from midostaurin. Gilteritinib In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib. Ensidipine In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg TM ) or tariline-vadatuximab (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor. In some embodiments, the additional agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0177] In some embodiments, the method of treatment further comprises the step of treating the subject with a chemotherapeutic agent or hematopoietic stem cell before administering the ABP, the pharmaceutical composition, or the immune response cell. In some embodiments, the method of treatment further comprises the step of treating the subject with a chemotherapeutic agent or hematopoietic stem cell after administering the ABP, the pharmaceutical composition, or the immune response cell. In some embodiments, the administering step is not followed by or is not performed in combination with immunoglobulin therapy. In some embodiments, the immunoglobulin therapy is intravenous immunoglobulin (IVIG) therapy.

[0178] In some embodiments, the subject has a refractory disease. In some embodiments, the subject has a relapsed disease. In some embodiments, the subject is an adult AML patient. In some embodiments, the subject is a pediatric AML patient.

[0179] In some embodiments, the subject has been administered a chemotherapeutic agent or has undergone hematopoietic stem cell therapy prior to administering a therapeutically effective amount of an ABP, pharmaceutical composition, or immune response cell to the subject.

[0180] In some embodiments, the subject is unresponsive to chemotherapy or hematopoietic stem cell therapy.

[0181] In some embodiments, the subject suffers from myeloblastic (M0) type AML. In some embodiments, the subject suffers from myeloblastic (M1) type AML. In some embodiments, the subject suffers from myeloblastic (M2) type AML. In some embodiments, the subject suffers from promyelocytic (M3) type AML. In some embodiments, the subject suffers from myelomonocytic (M4) type AML. In some embodiments, the subject suffers from monocytic (M5) type AML. In some embodiments, the subject suffers from erythroleukemia (M6) type AML. In some embodiments, the subject suffers from megakaryocyte (M7) type AML.

[0182] In some embodiments, the method of treatment includes administering an effective amount of immune response cells comprising a CAR targeting CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, the effective amount is a dose of 100,000 cells / kg to 25 million cells / kg of immune response cells. In some embodiments, the effective amount is a dose within the range of 100,000 cells / kg to 25 million cells / kg. In some embodiments, the effective amount is a dose within the range of 100,000 cells / kg to 15 million cells / kg.

[0183] In one aspect, the present disclosure provides a bispecific T cell engager (BiTE) comprising an antigen binding domain and a T cell activation domain, wherein the antigen binding domain specifically binds to a target protein / antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

[0184] In some embodiments, the antigen binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to the target protein.

[0185] In some embodiments, the T cell activation domain includes the intracellular domain of CD3ζ.

[0186] In some embodiments, the T cell activation domain comprises a single chain variable fragment (scFV) of an antibody that specifically binds CD3.

[0187] In another aspect, the present disclosure provides a polynucleotide encoding a BiTE. In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding a BiTE.

[0188] In another aspect, the present disclosure provides a method of treating a subject having a myeloid disorder (MD) or acute leukemia (AL), the method comprising: administering a therapeutically effective amount of a BiTE.

[0189] In some embodiments, myeloid disorders (MD) and acute leukemias (AL) are pediatric or adult-onset.

[0190] In some embodiments, the BiTE or vector is administered in combination with an additional agent.

[0191] In some embodiments, the additional agent is a chemotherapeutic agent or a biologic agent. In some embodiments, the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacitidine, decitabine, and CPX-351 In some embodiments, the additional agent is a Hedgehog signaling pathway inhibitor. In some embodiments, the Hedgehog signaling pathway inhibitor is a Sonic Hedgehog signaling pathway inhibitor. In some embodiments, the Sonic Hedgehog signaling pathway inhibitor is selected from Vismodegib, Sonidegib, and arsenic trioxide (ATO). In some embodiments, the Hedgehog signaling pathway inhibitor is Daurismo TM In some embodiments, the additional agent is a FMS-like tyrosine kinase 3 (FLT3) inhibitor. In some embodiments, the FLT3 inhibitor is selected from midostaurin. Gilteritinib In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In some embodiments, the IDH1 or IDH2 inhibitor is ivosidenib. Ensidipine In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the additional agent is a CD33 targeting agent. In some embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (MylotargTM ) or tariline-vadatuximab (SGN-CD33A). In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor. In some embodiments, the additional agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody. 2. Description of the drawings

[0192] These and other features, aspects and advantages of the present disclosure will become better understood through the following description and accompanying drawings, in which:

[0193] Figure 1 is a flow chart illustrating the method for identifying novel AML TSAs described in Example 1. The screening flow chart shows the original 2 gene lists selected from the AML gene expression data at diagnosis, and the priorities determined according to specific inclusion and exclusion criteria, defining the expression of genes in AML cell lines (SHI-1, HL-60, MOLM-13, MV4;11 and Kasumi-1) and healthy control hematopoietic cells (classified as CD3 + 、CD19 + and CD33 + Subpopulations of PBMC and CD34 isolated from cord blood + 26 / 76 genes were analyzed by flow cytometry in AML cells. The best candidate TSA was confirmed to be localized on the surface of AML cells by immunofluorescence.

[0194] Figure 2A Data from flow cytometric analysis of several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13, and MV4-11) using two different CD69 antibodies (light gray histograms) and subtypes (dark gray histograms) are shown. Subtype cytometric analysis data are provided as controls (dark gray histograms). Subtype controls are antibodies that retain similar properties to the primary antibody but lack specific target binding. Figure 2B Shown are images of fluorescent immunohistochemistry of an AML cell line (SHI-1) using two CD69 antibodies (white dots) as well as membrane staining (Membrite: colocalization of CD69 with a plasma membrane-specific dye). Figure 2C Shown are flow cytometry data using the CD69 antibody to analyze healthy hematopoietic cells (ie, healthy CD3, CD19, and CD33 subsets from PBMCs and CD34 positive cells from cord blood).

[0195] Figure 3A Data from flow cytometric analysis of several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) using two different CD63 antibodies (light grey histograms) are shown. Data from cytometric analysis of subtypes serve as controls (dark grey histograms). Figure 3B Shown are images of an AML cell line (HL-60) subjected to fluorescent immunohistochemistry using two CD63 antibodies (white dots) and membrane staining (Membrite: colocalization of CD63 with a plasma membrane-specific dye). Figure 3C Data are shown for flow cytometric analysis of healthy hematopoietic cells (healthy CD3, CD19 and CD33 subsets derived from PBMCs and CD34 positive cells from cord blood) using CD63 antibody.

[0196] Figure 4A Data from flow cytometric analysis of several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) using two different CD151 antibodies (light grey histograms) are shown. Data from cytometric analysis of subtypes serve as controls (dark grey histograms). Figure 4B Shown are images of fluorescent immunohistochemistry of an AML cell line (HL-60) using two CD151 antibodies (white dots) together with membrane staining (Membrite: co-localization of CD151 with a plasma membrane-specific dye). Figure 4C Data are shown for flow cytometric analysis of healthy hematopoietic cells (healthy CD3, CD19 and CD33 subsets derived from PBMCs and CD34 positive cells from cord blood) using CD151 antibody.

[0197] Figure 5A Data from flow cytometric analysis of several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13 and MV4-11) using two different CD84 antibodies (light grey histograms) are shown. Data from cytometric analysis of subtypes serve as controls (dark grey histograms). Figure 5B Shown are images of an AML cell line (HL-60) subjected to fluorescent immunohistochemistry using two CD84 antibodies (white dots) and membrane staining (Membrite: colocalization of CD84 with a plasma membrane-specific dye). Figure 5C Data are shown for flow cytometric analysis of healthy hematopoietic cells (healthy CD3, CD19 and CD33 subsets derived from PBMCs and CD34 positive cells from cord blood) using the CD84 antibody.

[0198] Fig. 6AData from flow cytometric analysis of several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13, and MV4-11) using two different CD109 antibodies (light grey histograms) are shown. Data from cytometric analysis of subtypes serve as controls (dark grey histograms). Figure 6B Shown are images of AML cell lines (Kasumi-1 or HL-60) subjected to fluorescent immunohistochemistry using two CD109 antibodies (white dots) and membrane staining (Membrite: colocalization of CD109 with a plasma membrane-specific dye). Figure 6C Data are shown using flow cytometric analysis of healthy hematopoietic cells (healthy CD3, CD19 and CD33 subsets derived from PBMCs and CD34 positive cells from cord blood) using the CD109 antibody.

[0199] Fig. 7A Data from flow cytometric analysis of several AML cell lines (HL-60, SHI-1, KASUM-1, MOLM-13, and MV4-11) using two different CD72 antibodies (light grey histograms) are shown. Data from cytometric analysis of subtypes served as controls (dark grey histograms). Figure 7B Images of an AML cell line (SHI-1) subjected to fluorescent immunohistochemistry using two CD72 antibodies (white dots) as well as membrane staining (Membrite: colocalization of CD72 with a plasma membrane-specific dye) are shown. Figure 7C Data are shown of flow cytometric analysis of healthy hematopoietic cells (healthy CD3, CD19 and CD33 subsets derived from PBMCs and CD34 positive cells from umbilical cord blood) using the CD72 antibody.

[0200] Figure 8 Shown are the results of flow cytometric analysis of human primary skin fibroblasts used to test the specificity of TSA.

[0201] Fig. 9 Results of flow cytometric analysis of different cancer cell lines used to test TSA specificity are shown. TSA expression levels were assessed by flow cytometric analysis in cell lines from different cancer types and are shown as histograms (light grey) versus subtype controls (grey).

[0202] Fig.10 Shown are the results of flow cytometric analysis of AML cell lines using different commercial antibodies.

[0203] Fig.11Figure 2 Expression of TSA mRNA in AML samples collected at diagnosis and in remission after treatment is shown. TSA mRNA expression was assessed by quantitative PCR in 14 paired pediatric AML bone marrow samples collected at diagnosis (RQ = 1) and in remission after treatment (dots represent mRNA expression values ​​in remission relative to diagnosis). Patients were stratified by AML genetic risk (SR = standard risk; HR = high risk). Despite differences in TSA mRNA expression, a significant decrease in CD69, CD63, and CD84 mRNA expression was found after treatment (* indicates p < 0.05) (RQ < 1).

[0204] Figures 12A-12B TSA expression in the clinic by flow cytometry analysis is shown. Fig. 12A Shown on CD34 + The CD38- subset lacks CD69 and CD84 expression. Fig. 12B Shown is TSA expression in AML samples at diagnosis, showing the highest expression of CD69 and CD84 in AML blasts.

[0205] Fig.13 Pipeline for the selection and construction of novel AMLCARs for CD69 and CD84. Ten scFv sequences were selected from phage display and prioritized based on specificity for expression in CAR expression cassettes cloned into generation 3 lentiviral transfer plasmids.

[0206] Fig.14 . CAR-T cell preparation workflow. Schematic diagram of the lentiviral transduction process and CAR-T cell preparation. The workflow includes the isolation of donor T cells on day 1, efficient activation with TransAct, and gene transfer of the CAR-LV construct on day 3. Starting from day 4, CAR-T cells (using scFv sequences B8 or F12 to produce CD84 and G1 or H3 to produce CD69) were expanded in TexMACS medium supplemented with IL-7 and IL-15 for 14 days. On day 17, CAR-T cells were immunophenotyped and co-cultured with HL-60 or SHI-1 (CD84+CD69+) AML cell lines.

[0207] Fig.15 Transduction efficiency was determined at day 17 by measuring vector copy number (VCN, # of integrated viral copies per cell). VCN was determined by digital droplet PCR (ddPCR) analysis with primers for the lentiviral backbone of all CAR-T cell constructs in each experiment. Each symbol represents a different CAR construct (CD84 produced with scFv sequences B8 or F12, CD69 produced with G1 or H3, and mock transduction [empty CAR]).

[0208] Fig.16 Flow cytometric analysis of cell surface expression of CD69 and CD84 on the indicated AML cell lines and subtype controls (dark grey peaks). Representative histograms of 3 independent experiments.

[0209] Fig.17 . Representative fold expansion kinetics of T cells during CAR-T cell preparation (from day 0 to day 14). Mock-transduced (empty CAR) and T cells were monitored, and cell number was calculated by flow cytometry. Data are expressed as mean ± SEM (n = 2 to 7, P>0.05). Each symbol represents a different CAR construct (CD84 produced with scFv sequence B8 or F12, CD69 produced with G1 or H3, and mock transduction [empty CAR]).

[0210] Figures 18A-18B At the end of preparation (day 14), the frequency of live T lymphocytes in lymphocytes was measured by flow cytometry ( Fig.18A ) and CD4 + and CD8 + The percentage of cells ( Fig.18B ). Bar graphs and symbols represent different CAR-T cell products (CD84 produced with scFv sequences B8 or F12, CD69 produced with G1 or H3, and mock transduction [empty CAR]). Data are presented as mean ± SEM of 2-5 different experiments.

[0211] Figures 19A-19B Representative flow cytometry analysis from day 1 to the end of CAR-T cell preparation (day 14) ( Fig.19A ) and the frequencies of T cell subsets ( Fig.19B ). Immunophenotype was determined by flow cytometry: natural (T n ) and stem cell memory (T scm ), CD197(CCR7) + CD45RO-; central memory (T cm )、CD197(CCR7)+CD45RO+; terminal differentiation (T ef )、CD197(CCR7)-CD45RO-;Effect memory (T em ) and transition memory (T tm ), CD197(CCR7)-CD45RO+. The bar graph represents different CAR-T cell products (generated with scFv sequences B8 or F12 against CD84, and G1 or H3 against CD69, and mock transduction [empty CAR]).

[0212] Figures 20A-20BOn days 1 and 17, the immunophenotype of CAR-T cells was determined by flow cytometry to evaluate their activation by the expression of the indicated markers ( Fig. 20A ) and depletion ( Fig. 20B ). Data represent mean ± SEM of 1-4 different experiments. Bar graphs represent different CAR-T cell products (generated with scFv sequences B8 or F12 against CD84, and G1 or H3 against CD69, and mock transduction [empty CAR]).

[0213] Fig.21 T cells engineered to express CD84 (F12, B8) CAR and CD69 (G1, H3) CAR recognized and killed target AML cell lines. + CD69 + ) AML cell lines were co-cultured with TexMACS medium (--) or effector CAR-T cells at a 1:1 E:T ratio for 48 h. Target AML cell lines stained with 7AAD and Annexin-V were analyzed by flow cytometry (by CD33 + Monitoring). When co-cultured with CAR-T cells, SHI-1 and HL-60 cell lines had a higher proportion of cell death than when co-cultured with mock-transduced cells (empty CAR) (*P<0.05, **P<0.01 vs. empty CAR). The bar graph represents different CAR-T cell products (CD84 produced with scFv sequences B8 or F12, CD69 produced with G1 or H3, and empty CAR). The data represent the mean ± SEM of 1-4 different experiments.

[0214] Fig. 22 . Each column represents the percentage of killing (% of lysis efficacy) exerted by different CAR-T products on the target AML cell line. The bar graph represents different CAR-T cell products (generated with scFv sequences B8 or F12 for CD84, and G1 or H3 for CD69, and mock transduction [empty CAR]). The data are expressed as the mean ± SEM of 1-3 different experiments (*P<0.05 vs. empty CAR). The CAR-T cell lysis titer is calculated as follows:

[0215]

[0216] Fig.23. The percentage of CAR-T cell lysis measured by bioluminescence (BLI) in the target AML cell line transduced with luciferase (LUC). Each column represents the percentage of killing imposed by different CAR-T products on the AML-LUC cell line, standardized with respect to the BLI value of the relative AML cell line cultured alone. The bar graph represents different CAR-T cell products (generated with scFv sequence B8 or F12 for CD84, and G1 or H3 for CD69, and simulated transduction [empty CAR]). Data are expressed as mean ± SEM of 2 different experiments (***P<0.001; ****P<0.0001vs. empty CAR).

[0217] Fig.24 . Live CAR-T cells were cultured with target AML cell lines alone (-) or at a 1:1 effector:target (E:T) ratio for 48 hours, and the absolute numbers were quantified by flow cytometry. The data show the persistence of effector cells and the lysis of target AML cells. The dashed line represents CAR-T cells at the first 17 days of coculture. Each symbol represents a different CAR-T cell product (CD84 produced with scFv sequences B8 or F12, CD69 produced with G1 or H3, and mock transduced [empty CAR]) and cell line.

[0218] Figures 25A-25B . ( Fig.25A ) representative images and ( Fig.25B ) from CD34 + The absolute number of colony forming units (CFU) produced by cells, the CD34 + Cells were cultured alone (---) or with CAR-T cells at a 1:1 E:T ratio for 6 hours and then plated in MethoCult medium for 14 days. Bars and symbols represent different CAR-T cell products (CD84 produced with scFv sequences B8 or F12, CD69 produced with G1 or H3, and mock transduced [empty CAR]) and human CD34 + Cells. Data are presented as mean ± SEM of 2-3 different experiments (ns, p>0.05 not significant).

[0219] Figures 26A-26C . Luciferase bioluminescence signal observed in leukemic mouse xenografts Fig.26A It shows that CD69 (H3) CAR-T cells can inhibit CD69 in leukemia mouse xenografts. + Effects on the growth of AML cell lines. Fig.26B The in vivo lytic efficacy of CAR-T cells against the corresponding AML cell lines, represented by the reduction in luciferase signal (expressed as total flux), is provided (*P<0.05 vs. mock-transduced cells [empty CAR]). Fig.26C The in vivo lytic efficacy of CD69 (H3 and G1) and CD84 (B8 and F12) CAR-T cells on CD69+CD84+ AML cell lines is shown (*P<0.05 relative to empty CAR). NSG mice were injected with 0.5×10 6 SHI-1-LUC cells were injected 2 days later with 1.5 × 10 6 CAR-T (1:3). N = 7 animals / group). Data are presented as mean ± SEM. AML engraftment and spread were monitored weekly by luciferase bioluminescence.

[0220] Figures 27A-27D The in vivo efficacy of B8 and F12 scFv chains against CD84 and H3ScFv against CD69 is provided. The in vivo lytic efficacy of the corresponding CAR-T cells against the SHI-1 target AML cell line is indicated by the reduction of luciferase signal (expressed as total flux). (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001, Mann Whitney test). NSG mice were injected with 0.5×10 6 SHI-1-luciferase positive cells were injected 2 days later with 1.5 × 10 6 CAR-T (1:3 ratio. N = 3-7 animals / group). + CD69 + )AML cell lines and ( Fig.27C )F12 / B8 or empty CAR-T cells (no scFv) and ( Fig.27D ) Survival of NSG mice with either CAR-T cells or empty CAR-T cells. Data are presented as mean ± SEM. AML engraftment and spread were monitored weekly by luciferase bioluminescence; *p < 0.05, Mantel-Cox test.

[0221] Figures 28A-28B The in vivo specificity of B8 and F12 scFv chains against CD84 was provided. Anti-CD84 (B8) CAR-T cell effects ( Fig.28A )CD84 neg K562 and ( Fig.28B )CD84 neg U937 AML cell line. The in vivo lytic efficacy of CAR-T cells against non-target AML cell lines was indicated by the reduction of luciferase signal (expressed as total flux). NSG mice were injected with 0.5×10 6 AML-LUC + Cells were injected 2 days later with 1.5×10 6CAR-T (ratio of 1:3. N=6-10 animals / group). Data are presented as mean±SEM. AML engraftment and spread were monitored weekly by luciferase bioluminescence.

[0222] Fig.29 Provided is the expression of new TSAs in primary AML cells derived from patient-derived xenografts (AML-PDX). The median fluorescence intensity (MFI) of CD84, CD69, and CD72 expression on the surface of AML cells collected from PDX models generated from AML samples of children newly diagnosed with AML was detected by flow cytometry (subtype control, left peak-dark gray; AML, right peak-light gray). Representative histograms of 2 independent experiments.

[0223] Fig.30 Provides the in vitro lytic efficacy of anti-CD84 CAR-T cells on primary AML cells. When CAR T cells are co-cultured with target primary ex vivo cells collected from the AML-PDX model at an effector: target ratio of 1: 1, 48 hours), the percentage of AML cells killed by the generated CAR T cells expressing anti-CD84 (PDX#5 does not include B8) ScFv. Analyze ex vivo target AML primary cells stained with 7AAD and Annexin-V (monitored by CD33 expression) by flow cytometry (the lytic efficacy has been standardized for the lytic efficacy induced by empty CAR T cells).

[0224] Figures 31A-31B The in vitro lytic efficacy of anti-CD69 A1, F1, C2, H2 ScFv is provided. Anti-CD69 A1, F1, C2, H2 ScFv and target cells ( Fig.31A )SHI-1 and ( Fig.31B )After 48 hours of co-culture of HL60AMLCD84+CD69+ cell lines, the lytic efficacy induced by anti-CD69A1, F1, C2, H2ScFv, and the expression of 7AAD and Annexin-V was measured by flow cytometry. Each bar graph represents the percentage of killing effect of different CAR-T cell products on cell lines, which is normalized compared with the cell lines cultured with CAR-T cells alone (---) and empty CAR T cells.

[0225] Figures 32A-32B The in vitro cytokine production capacity of anti-CD84 B8, F12 ScFv(s)CAR T cells was shown. + / CD69 + ) AML cell lines were co-cultured at a 1:1E:T ratio for 48 hours, as measured by flow cytometry ( Fig.32A)IFNγ and ( Fig.32B ) Percentage of TNFα-positive cells Each bar represents the mean ± SEM of 1-4 independent experiments. (*p<0.05, **p<0.005, ***p<0.0005, MannWhitney T-Test).

[0226] Figures 33A-33B Shown when with CD34 + In vitro off-target cytotoxicity results of B8 and F12 scFv chains against CD84 ScFv in co-culture with HSC. + The absolute number of total colony-forming units (CFU) generated by HSC alone (---) or with CAR-T cells at a 1:1 E:T ratio for 6 hours and then cultured in MethoCult medium for 12 days ( Fig.33A ) and colony morphology ( Fig.34B ) to distinguish multipotent granulocyte, erythroid, macrophage and megakaryocyte colonies, granulocyte-macrophage colonies, granulocyte and macrophage colonies (CFU-GEMM, CFU-GM, CFU-G and CFU-M, respectively) (data are expressed as mean ± SEM, n = 1-2, p > 0.05 not significant).

[0227] Figures 34A-34B The in vivo specificity of the B8 and F12 scFv chains against CD84 was provided. Anti-CD84 (B8) CAR-T cells against SHI-1-CD84 ko Role of AML cell lines ( Fig.34A The in vivo lytic efficacy of CAR-T cells was indicated by the reduction of luciferase signal (expressed as total flux). 6 AML-LUC + SHI-1 ko The cells were injected into NSG mice and on days 2 and 8, 1.5 × 10 6 CAR-T (E:T 1:3, n = 4 animals / group). Data are presented as mean ± SEM. SHI-1 was monitored weekly by luciferase bioluminescence. ko Transplantation and spread. Fig.34B ) injection of SHI-1-CD84 ko Survival rate of NSG mice treated with AML cell lines and F12 / B8 or empty CAR-T cells. (Data are expressed as mean ± SEM, p>0.05 is not significant).

[0228] Figures 35A-35BProvides in vitro killing capacity of B8 CD84 and F12 CD84 CAR-T cells and A1 CD69C2 CD68 CAR-T cells. When co-cultured with target primary ex vivo cells collected from AML-PDX models, the cells produced by Fig.35A ) The percentage of AML cells killed by CAR T cells with anti-CD84 (B8 and F12) or anti-CD69 (A1 and C2) scFv chains (E: T ratio 1: 1, 48 hours). The histogram shows cell death of AML primary cells (monitored by CD33 expression and stained with 7AAD and Annexin-V) by flow cytometry. The lytic potency was normalized to the lytic potency induced by empty CAR T cells. Each histogram represents the mean ± SEM of each replicate, n = 1-2 replicates per group).

[0229] Figures 36A-36B The in vitro cytokine production capacity of anti-CD84B8, F12 scFv was provided. + After AML primary cells were co-cultured at a 1:1 E:T ratio for 48 hours, the CAR T cells ( Fig.36A )IFNγ and ( Fig.36B )TNFα production. Data were normalized to cytokine production by CAR T cells in medium alone (---). Representative experiment (p>0.05 not significant).

[0230] Figures 37A-37C In vivo efficacy of the B8 ScFv chain against AML-PDX expressing CD84 is provided. Fig.37A ) NSG mice were injected with 1.0×10 6 AML-luciferase (LUC) expressing cells were used to monitor AML engraftment and spread weekly by LUC bioluminescence (expressed as total flux). On day +28 from AML injection, LUC analysis yielded a reliable signal of AML engraftment, and mice were tail-injected with 5×10 6 CAR-T cells (E:T 5:1 ratio) or 10×10 6 CAR-T cells (E:T 10:1 ratio, n = 5-10 animals / group). Figures 37B-37C ) Day 7 after CAR-T cell inoculation ( Fig.37B ) or Day 12 ( Fig.37C Frequency distribution of CAR T cells (% of CD3+ lymphocytes) and PDX-AML cells in peripheral blood (PB), bone marrow (BM), and spleen (SPL) of mice injected with AML-PDX and CAR-T cells on day 16. (nd = not detected, data are expressed as mean ± SEM of 1-2 mice per group).

[0231] Figures 38A-38B In vivo efficacy of the B8 ScFv chain against AML-PDX cells expressing CD84 is provided. Fig.38A )NSG mice were injected with 1.0×10 6 AML-luciferase (LUC) expressing cells were cultured 2 days later with 5×10 6 CAR-T cells (E:T 10:1) or 10×10 6 CAR-T cells (E:T 10:1 ratio, n = 5-10 animals / group) treatment. Fig.38B ) Frequency distribution of lymphocytes and PDX-AML cells in the peripheral blood (PB), bone marrow (BM), spleen (SPL), and lung (LNG) of mice injected with AML-PDX and CAR-T cells on day 12 after CAR-T vaccination (data are expressed as mean ± SEM of 1-2 mice per group).

[0232] Figures 39A-39H In vivo off-target effects of the B8 ScFv chain on hematopoietic precursors are provided. Fig.39A ) After sublethal irradiation, 10 6 Personal CD34 + NSG mice were transplanted with hematopoietic stem cells (HSCs) and human leukocyte engraftment was examined weekly by flow cytometry. 6 Mice were tail-injected intravenously with empty cells or anti-CD84 B8 CAR T cells, and changes in peripheral blood leukocyte composition were monitored daily from day +1 to day +8 after infusion. Fig.39B ) is used to monitor the transplantation of hCD34 + Representative flow cytometric analysis strategy of HSC-derived leukocyte engraftment in mouse peripheral blood. Figures 39C-39H ) Flow cytometric analysis was used to identify changes in the frequency of human hematopoietic precursors in the peripheral blood of mice after infusion of empty CAR or B8 CAR T cells (n=1).

[0233] Figures 40A-40K Showing CAR binder (ScFv) and CD69 ( Figures 40A-40H ) or CD84( Figures 40I-40K ) Molecular models of ScFv in complex with the extracellular domain of CD69 or CD84 (sequences provided by uniprot) were generated by ChimeraX software (v.1.5); contact residues are shown and highlighted in the sequence. Figures 40A-40HThe predicted structures of the extracellular domain of CD69 bound to scFv A1, C1, F1, G1, C2, F2, H2 or H3 are shown (upper panel), and the amino acid sequences of the scFvs with the binding domains highlighted (lower panel). The results show that scFv A1, C1, F1, G1, C2, F2, H2 and H3 bind similarly to the extracellular domain of CD69. The amino acid chain docking between scFv A1, C1, F1, G1, C2, F2, H2 and H3 is similar and spans the entire extracellular domain of CD69 and scFv. Figures 40I-40K The predicted structure of the CD84 extracellular domain bound to scFv B8 and F12 (upper panel) and the amino acid sequence of the scFv with the binding domain highlighted (lower panel) are shown. These results indicate that scFv B8 and F12 bind similarly to the extracellular domain of CD84. The amino acid chain docking between scFv B8 and F12 is similar and spans the entire extracellular domain of CD84 as well as the scFv. 3. Specific implementation plan

[0234] 3.1 Definition

[0235] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms should include plural, and plural terms should include singular. Typically, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out according to the manufacturer's instructions, as generally completed in the art or as described herein. The terms and laboratory procedures and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry as described herein are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparations, preparations, and delivery and the treatment of patients.

[0236] Unless otherwise indicated, the following terms shall be understood to have the following meanings.

[0237] Unless further modified by the designation of a non-human species, the terms "CD63", "CD63 protein" and "CD63 antigen" are used interchangeably herein to refer to human CD63, or any variant (e.g., splice variants and allelic variants), isoforms or species orthologs of human CD63 that are naturally expressed by cells (including tumor cells) or expressed by cells transfected with the CD63 gene (NCBI Accession No.: NG_008347, gene ID 967).

[0238] Unless further modified by the designation of a non-human species, the terms "CD151", "CD151 protein" and "CD151 antigen" are used interchangeably herein to refer to human CD151, or any variants (e.g., splice variants and allelic variants), isoforms or species orthologs of human CD151 that are naturally expressed by cells (including neoplastic cells) or expressed by the CD151 gene (NCBI Accession No.: NG_007478.1, gene ID 977).

[0239] Unless further modified by the nomenclature of a non-human species, the terms "CD72," "CD72 protein," and "CD72 antigen" are used interchangeably herein to refer to human CD72, or any variants (e.g., splice variants and allelic variants), isoforms or species orthologs of human CD72 that are naturally expressed by cells (including tumor cells), or expressed by cells expressing the CD72 gene (NCBI Accession No.: NC_000009.12, gene ID 971).

[0240] Unless further modified by the designation of a non-human species, the terms "CD84," "CD84 protein," and "CD84 antigen" are used interchangeably herein to refer to human CD84, or any variant (e.g., splice variants and allelic variants), isoforms or species orthologs of human CD84 that are naturally expressed by cells (including tumor cells) or expressed by cells transfected with the CD84 gene (NCBI Accession No.: NC_000001.11, gene ID 8832).

[0241] Unless further modified by the designation of a non-human species, the terms "CD69," "CD69 protein," and "CD69 antigen" are used interchangeably herein to refer to human CD69, or any variant (e.g., splice variants and allelic variants), isoforms or species orthologs of human CD69 that are naturally expressed by cells (including tumor cells), or expressed by cells expressing the CD69 gene (NCBI Accession No.: NC_000012.12, gene ID 969).

[0242] Unless further modified by the designation of a non-human species, the terms "CD109", "CD109 protein" and "CD109 antigen" are used interchangeably herein to refer to human CD69, or any variant (e.g., splice variants and allelic variants), isoforms or species orthologs of human CD109 that are naturally expressed by cells (including tumor cells) or expressed by the CD109 gene (NCBI Accession No.: NC_000006.12, gene ID 135228).

[0243] The term "antigen binding protein" (ABP) refers to a protein comprising one or more antigen binding domains that specifically bind to an antigen or epitope. In some embodiments, the antigen binding domain binds to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP consists of an antibody. In some embodiments, the ABP consists essentially of an antibody. In some embodiments, the ABP comprises an alternative scaffold. In some embodiments, the ABP consists of an alternative scaffold. In some embodiments, the ABP consists essentially of an alternative scaffold. In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP consists of an antibody fragment. In some embodiments, the ABP consists essentially of an antibody fragment. For example, as provided herein, "CD63", "anti-CD63 ABP" or "CD63-specific ABP" is an ABP that specifically binds to the antigen CD63. In some embodiments, the ABP binds to the extracellular domain of CD63, CD151, CD72, CD84, CD69 or CD109. In certain embodiments, a CD63, CD151, CD72, CD84, CD69, or CD109 ABP provided herein binds to an epitope of CD151, CD72, CD84, CD69, or CD109, respectively, that is conserved between or among CD151, CD72, CD84, CD69, or CD109 proteins from different species.

[0244] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen binding domains that specifically bind to an antigen or epitope. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen binding domain is a V-binding domain. H -V L The antigen binding domain formed by dimerization. Antibodies are a type of ABP.

[0245] The term "antigen binding domain" refers to the portion of an ABP that is capable of specifically binding to an antigen or epitope.

[0246] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody that has a structure substantially similar to a naturally occurring antibody structure and has heavy chains that include an Fc region.

[0247] The term "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain that interacts with Fc receptors and certain proteins of the complement system in naturally occurring antibodies. The structures of the Fc regions of various immunoglobulins and the glycosylation sites contained therein are known in the art. See Schroeder and Cavacini, J. Allergy Clin. Immunol., 2010, 125: S41-52, which is incorporated herein by reference in its entirety. The Fc region may be a naturally occurring Fc region, or a modified Fc region as described elsewhere in this disclosure.

[0248] "Antibody fragments" include a portion of an intact antibody, such as the antigen binding region or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0249] An "Fv" fragment comprises a non-covalently linked dimer of one heavy- and one light-chain variable domain.

[0250] "Fab" fragments contain, in addition to the heavy and light chain variable regions, the constant region of the light chain and the first constant region (CH1) of the heavy chain. Fab fragments can be produced, for example, by recombinant methods or by papain digestion of full-length antibodies.

[0251] "F(ab')2" fragments contain two Fab' fragments connected by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by recombinant methods or by pepsin digestion of intact antibodies. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0252] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain V H Domain and V L Domain. V H and V LTypically linked by a peptide linker. See Plückthun A. (1994). In some embodiments, the linker is (GGGGS)n (SEQ ID NO: 1). In some embodiments, n=1, 2, 3, 4, 5 or 6. See Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, which is incorporated herein by reference in its entirety.

[0253] "scFv-Fc" fragments comprise an scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. The Fc domain can be located within the V H or V L Then, depending on the orientation of the variable domains in the scFv (i.e., V H -V L or V L -V H ). Any suitable Fc domain known in the art or described herein can be used. In some cases, the Fc domain comprises an IgG4 Fc domain.

[0254] The term "single domain antibody" refers to molecules in which one variable domain of the antibody specifically binds an antigen without the presence of another variable domain.

[0255] A "monospecific ABP" is an ABP that contains a binding site that specifically binds a single epitope. An example of a monospecific ABP is the naturally occurring IgG molecule, which, although bivalent, recognizes the same epitope in each antigen binding domain. The binding specificity may be present in any suitable valency.

[0256] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous antibody population. A substantially homogeneous antibody population comprises antibodies that are substantially similar and that bind to the same epitope, except for variants that may generally occur during the production of the monoclonal antibody. These variants generally exist only in small amounts. Monoclonal antibodies are generally obtained by methods that include selecting a single antibody from a plurality of antibodies. For example, a selection method may be to select a unique clone from a plurality of clones, such as a library of a hybridoma clone, a phage clone, a yeast clone, a bacterial clone, or other recombinant DNA clones. The selected antibody may be further altered, for example, to improve affinity to the target ("affinity maturation"), humanize the antibody, improve its yield in cell culture, and / or reduce its immunogenicity in a subject.

[0257] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0258] The "humanized" form of non-human antibodies is a chimeric antibody containing the minimum sequence derived from a non-human antibody. Humanized antibodies are usually human antibodies (receptor antibodies), wherein the residues from one or more CDRs are replaced by the residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as mice, rats, rabbits, chickens, camels or non-human primate antibodies with desired specificity, affinity or biological effects. In some cases, the selected framework region residues of the receptor antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies can also be included in residues not found in the receptor antibody or the donor antibody. These modifications can be performed to further improve antibody function.

[0259] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing a human antibody repertoire or human antibody encoding sequences (e.g., obtained from a human source or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0260] "Isolated ABP" or "isolated nucleic acid" is an ABP or nucleic acid that has been separated and / or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other protein or non-protein substances. In some embodiments, the isolated ABP is purified to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, for example, by using a spinning cup sequencer. In some embodiments, the isolated ABP is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or non-reducing conditions and detected by Coomassie blue or silver staining. The isolated ABP includes the ABP in situ within the recombinant cell because at least one component of the natural environment of the ABP is absent. In some embodiments, the isolated ABP or isolated nucleic acid is prepared by at least one purification step. In some embodiments, the isolated ABP or isolated nucleic acid is purified to, by weight, at least 80%, 85%, 90%, 95% or 99%. In some embodiments, the isolated ABP or isolated nucleic acid is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight of the ABP or nucleic acid. In some embodiments, the isolated ABP or isolated nucleic acid is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by volume of the ABP or nucleic acid.

[0261] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., ABP) and its binding partner (e.g., antigenic epitope). Unless otherwise indicated, as used herein, "affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., ABP and antigen or epitope). The affinity of a molecule X for its partner Y can be expressed by the dissociation equilibrium constant (K D ). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by conventional methods known in the art, including those described herein. For example, surface plasmon resonance (SPR) techniques (e.g., ) or biolayer interferometry (e.g. ), or by monoclonal competitive ELISA testing to determine affinity.

[0262] With respect to the binding of an ABP to a target molecule, the terms "bind," "specifically binds," "binds specifically to," "has specificity for," "selectively binds," and "has selectivity for" a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from non-specific or non-selective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In this case, specific binding is indicated if the binding of the ABP to the target molecule is competitively inhibited by the control molecule. In some embodiments, the affinity of the CD63, CD151, CD72, CD84, CD69, or CD109 ABP for a non-target molecule is less than about 50% of the affinity for CD63, CD151, CD72, CD84, CD69, or CD109, respectively. In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for non-target molecules is less than about 40% of the affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for non-target molecules is less than about 30% of the affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of CD63, CD151, CD72, CD84, CD69 or CD109 ABP for non-target molecules is less than about 20% of the affinity for CD63, CD151, CD72, CD84, CD69 or CD109, respectively. In some embodiments, the affinity of a CD63, CD151, CD72, CD84, CD69, or CD109 ABP for a non-target molecule is less than about 10% of the affinity for CD63, CD151, CD72, CD84, CD69, or CD109, respectively. In some embodiments, the affinity of a CD63, CD151, CD72, CD84, CD69, or CD109 ABP for a non-target molecule is less than about 1% of the affinity for CD63, CD151, CD72, CD84, CD69, or CD109, respectively. In some embodiments, the affinity of a CD63, CD151, CD72, CD84, CD69, or CD109 ABP for a non-target molecule is less than about 0.1% of the affinity for CD63, CD151, CD72, CD84, CD69, or CD109, respectively.

[0263] As used herein, the term "kd" (sec-1) refers to the dissociation rate constant for a particular ABP-antigen interaction. This value is also referred to as the koff value.

[0264] As used herein, the term "ka" (M-1 x sec-1) refers to the association rate constant for a particular ABP-antigen interaction. This value is also referred to as the kon value.

[0265] The term "K D “(M)” refers to the dissociation equilibrium constant for a specific ABP-antigen interaction. K D =kd / ka.

[0266] The term "K A "(M-1) refers to the association equilibrium constant of a specific ABP-antigen interaction. kA = ka / kd.

[0267] An "affinity matured" ABP is an ABP with one or more changes (e.g., in one or more CDRs or FRs) that result in improved affinity of the ABP for its antigen compared to a parent ABP without the changes. In one embodiment, the affinity matured ABP has nanomolar or picomolar affinity for the target antigen. Affinity matured ABPs can be produced using a variety of methods known in the art. For example, Marks et al. (Bio / Technology, 1992, 10:779-783, which is incorporated herein by reference) describes affinity matured ABPs produced by V H and V L Affinity maturation of domain shuffling. Random mutagenesis of CDR and / or framework residues is described, for example, by Barbas et al. (Proc. Nat. Acad. Sci. USA, 1994, 91: 3809-3813); Schier et al., Gene, 1995, 169: 147-155; Yelton et al., J. Immunol., 1995, 155: 1994-2004; Jackson et al., J. Immunol., 1995, 154: 3310-33199; and Hawkins et al, J. Mol. Biol., 1992, 226: 889-896, each of which is incorporated herein by reference in its entirety.

[0268] An "immunoconjugate" is an ABP conjugated to one or more heterologous molecules.

[0269] "Effector functions" refer to those biological activities mediated by the Fc region of an antibody, which activity may vary depending on the antibody subtype. Examples of antibody effector functions include C1q binding to activate complement-dependent cytotoxicity (CDC), Fc receptor binding to activate antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP).

[0270] When used herein in the context of two or more ABPs, the term "compete with" or "cross-compete" means that two or more ABPs compete for binding to an antigen (e.g., CD63, CD151, CD72, CD84, CD69, or CD109). In an exemplary assay, CD63, CD151, CD72, CD84, CD69, or CD109 is coated on a surface and contacted with a first CD63, CD151, CD72, CD84, CD69, or CD109 ABP, respectively, followed by the addition of a second CD63, CD151, CD72, CD84, CD69, or CD109 ABP. In another exemplary assay, a first CD63, CD151, CD72, CD84, CD69 or CD109 ABP is coated on a surface and contacted with CD63, CD151, CD72, CD84, CD69 or CD109, and then a second CD63, CD151, CD72, CD84, CD69 or CD109 ABP is added. If the presence of the first CD63, CD151, CD72, CD84, CD69 or CD109 ABP reduces the binding of the second CD63, CD151, CD72, CD84, CD69 or CD109 ABP in any one assay, then the ABPs compete. The term "competes with" also includes a combination of ABPs, in which one ABP reduces the binding of another ABP, but no competition is observed when the ABPs are added in the opposite order. However, in some embodiments, the first and second ABPs inhibit the binding of each other, regardless of the order in which they are added. In some embodiments, one ABP reduces the binding of another ABP to its antigen by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%. The skilled person can select the concentration of the antibody used in the competition assay based on the affinity of the ABP for CD63, CD151, CD72, CD84, CD69, or CD109 and the titer of the ABP. The assays described in this definition are illustrative, and the skilled person can utilize any suitable assay to determine whether antibodies compete with each other.Suitable assays are described, for example, in Cox et al., "Immunoassay Methods," in Assay Guidance Manual [Internet], Updated December 24, 2014 (www.ncbi.nlm.nih.gov / books / NBK92434 / ; accessed September 29, 2015); Silman et al., Cytometry, 2001, 44:30-37; and Finco et al., J. Pharm. Biomed. Anal., 2011, 54:351-358; each of which is incorporated herein by reference in its entirety.

[0271] The term "epitope" refers to a portion of an antigen that specifically binds to an ABP. An epitope is typically composed of surface accessible amino acid residues and / or sugar side chains, and may have specific three-dimensional structural features as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that the binding to the former rather than the latter may be lost in the presence of a denaturing solvent. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. The epitope to which an ABP binds can be determined using known techniques for epitope determination, such as testing the binding of an ABP to CD63, CD151, CD72, CD84, CD69 or CD109 variants with different point mutations, or to chimeric CD63, CD151, CD72, CD84, CD69 or CD109 variants.

[0272] The "percent identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps (if necessary) to obtain the maximum percentage of sequence identity. Alignment for determining the percentage of amino acid sequence identity can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0273] The term "treating" (and variations thereof, such as "treat" or "treatment") refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be used both preventively and during clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

[0274] The term "subject" as used herein refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject suffers from a disease or condition that can be treated with an ABP, ABP-drug conjugate, or immune response cell comprising a CAR provided herein. In some embodiments, the disease or condition is cancer.

[0275] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0276] The term "myeloid" as used herein includes all cells belonging to the granulocyte (i.e., neutrophil, eosinophil, basophil), monocyte / macrophage, erythrocyte, megakaryocyte and mast cell lineages. Myeloid malignancies are clonal diseases of hematopoietic stem or progenitor cells. These malignancies can be present in the bone marrow and peripheral blood. They can be caused by genetic and epigenetic changes that interfere with key processes such as self-renewal, proliferation and impaired differentiation.

[0277] 3.2 Other interpretative conventions

[0278] Ranges recited herein should be understood as shorthand for including all values ​​within the range including the stated endpoints. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0279] Unless otherwise stated, reference to a compound having one or more stereocenters refers to each stereoisomer and all combinations of stereoisomers thereof.

[0280] 3.3 Compositions targeting tumor-specific antigens

[0281] One aspect of the present disclosure relates to a protein that targets a tumor-specific antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109.

[0282] In some embodiments, the tumor-specific antigen is CD63. In some embodiments, CD63 is a protein encoded by the CD63 gene (12q13.2) (NCBI accession number: NG_008347, gene ID 967).

[0283] In some embodiments, the tumor-specific antigen is CD151. In some embodiments, CD151 is a protein encoded by the CD151 gene (11p15.5) (NCBI accession number: NG_007478.1, gene ID 977). Multiple alternative splicing transcript variants encoding the same protein of this gene have been described. Any splicing variant can be used in various embodiments.

[0284] In some embodiments, the tumor-specific antigen is CD72. In some embodiments, CD72 is a protein encoded by the CD72 gene (9p13.3) (NCBI accession number: NC_000009.12, gene ID 971).

[0285] In some embodiments, the tumor-specific antigen is CD84. In some embodiments, CD84 is a protein encoded by the CD84 gene (1q23.3) (NCBI accession number: NC_000001.11, gene ID 8832).

[0286] In some embodiments, the tumor-specific antigen is CD69. In some embodiments, CD69 is a protein encoded by the CD69 gene (12p13.31) (NCBI accession number: NC_000012.12, gene ID 969).

[0287] In some embodiments, the tumor-specific antigen is CD 109. In some embodiments, CD 109 is a protein encoded by the CD 109 gene (6q13) (NCBI accession number: NC_000006.12, gene ID 135228).

[0288] 3.3.1 Antigen Binding Protein (ABP)

[0289] In one aspect, the present disclosure provides an antigen binding protein (ABP) that specifically binds to CD63, CD151, CD72, CD84, CD69 or CD109.

[0290] In certain embodiments, the ABP specifically binds to CD63. In certain embodiments, the ABP specifically binds to CD151. In certain embodiments, the ABP specifically binds to CD72. In certain embodiments, the ABP specifically binds to CD84. In certain embodiments, the ABP specifically binds to CD69. In certain embodiments, the ABP specifically binds to CD109. In some embodiments, the ABP is an isolated antigen binding protein (ABP) that specifically binds to human CD63, CD151, CD72, CD84, CD69 or CD109. In certain embodiments, the ABP specifically binds to CD84 or CD69.

[0291] In some embodiments, the ABP comprises a human Fc.

[0292] In some embodiments, the ABP is human, humanized, or chimeric.

[0293] In specific embodiments, the ABP is monoclonal.

[0294] In some embodiments, when administered, the ABP is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the ABP bound to cell surface CD63, CD151, CD72, CD84, CD69 or CD109 affects antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, natural killer (NK) cells achieve ADCC through the binding of the Fc domain of the ABP to CD16 on the surface of the NK cells.

[0295] In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP comprises an immunoglobulin constant region. Antibody fragments can also be any synthetic or genetically engineered protein. For example, antibody fragments include isolated fragments consisting of the light chain variable region, "Fv" fragments consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules (scFv proteins) in which the light and heavy chain variable regions are connected by a peptide linker.

[0296] Another form of antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs (also called "minimal recognition units" or "hypervariable regions") can be covalently or non-covalently incorporated into a molecule to make it an antigen binding protein. CDRs can be obtained by constructing a polynucleotide encoding a target CDR. For example, such polynucleotides are prepared by synthesizing the variable region using the polymerase chain reaction with mRNA from antibody-producing cells as a template (see, e.g., Larrick et al., Methods: A Companion to Methods in Enzymology 2:106, 1991; Courtenay Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995); and Ward et al., "Genetic Manipulation and Expression of Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley Liss, Inc. 1995).

[0297] Thus, in one embodiment, the antibody fragment comprises at least one CDR as described herein. The binding agent may comprise at least two, three, four, five or six CDRs as described herein. The antibody fragment may also comprise at least one variable region domain of an antibody as described herein. The variable region domain may have any size or amino acid composition and typically comprises at least one CDR sequence for binding to human CD63, CD151, CD72, CD84, CD69 or CD109, such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which are specifically described herein and are adjacent to or in the framework of one or more framework sequences. In general, the variable (V) region domain may be an immunoglobulin heavy chain (V H ) and / or light chain (V L ) any suitable arrangement of variable domains. Thus, for example, the V region domain may be monomeric and be V H or V Ldomains, which are capable of independently binding to the target antigen with an affinity ranging from 1 nM to 1 pM. Alternatively, the V region domains may be dimers and contain V H V H 、V H V L or V L V L Dimer. The V region dimer contains at least one V H and at least one V L Chain, which may be non-covalently bound (hereinafter referred to as F V If desired, the chains can be covalently coupled directly (eg, via a disulfide bond between the two variable domains) or via a linker (eg, a peptide linker) to form a single-chain Fv (scFV).

[0298] The variable region domain can be any naturally occurring variable domain or an engineered version thereof. An engineered version refers to a variable region domain produced using recombinant DNA engineering techniques. Such an engineered version includes, for example, those produced by a specific antibody variable region by inserting, deleting or changing the amino acid sequence of a specific antibody or inserting, deleting or changing the amino acid sequence of a specific antibody. Specific examples include engineered variable region domains comprising at least one CDR from a first antibody and optionally one or more framework amino acids and the remainder of the variable region domain from a second antibody.

[0299] The variable region domain may be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the V H The V domain can be linked to an immunoglobulin CH1 domain or a fragment thereof. L The domain may be linked to the CK domain or a fragment thereof. Thus, for example, the antibody may be a Fab fragment in which the antigen binding domain contains the relevant V domains covalently linked at their C-termini to the CH1 and CK domains, respectively. H and V L Domains. The CH1 domain may be extended with additional amino acids, for example to provide a hinge region or a portion of a hinge region domain as found in a Fab' fragment, or to provide additional domains, such as antibody CH2 and CH3 domains.

[0300] As described herein, the antibody comprises at least one of these CDRs. For example, one or more CDRs can be incorporated into known antibody framework regions (IgG1, IgG2, etc.), or conjugated to a suitable carrier to increase its half-life. Suitable carriers include, but are not limited to, Fc, polyethylene glycol (PEG), albumin, transferrin, etc. These and other suitable carriers are known in the art. This conjugated CDR peptide can be a monomer, dimer, tetramer or other form. In one embodiment, one or more water-soluble polymers are bonded to one or more specific positions of the binding agent, such as the amino terminus.

[0301] ABPs of the present disclosure that specifically bind to CD84 or CD69 can be found in Table 10 (V L and V H CDR) and Table 11 (light and heavy chain variable regions).

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] In some embodiments, the ABP comprises a light chain variable domain (V L )CDR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NO:45-51 of Table 10.

[0310] In some embodiments, the ABP comprises a light chain variable domain (V L ) CDR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NO:52-54 of Table 10.

[0311] In some embodiments, the ABP comprises a light chain variable domain (V L)CDR3, which comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NO:55-61 of Table 10.

[0312] In some embodiments, the ABP comprises: (a) V L CDR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 45-51 of Table 10; (b) V L CDR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 52-54 of Table 10; and (c) V L A CDR3 comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO:55-61 of Table 10.

[0313] In some embodiments, the ABP comprises: (a) V L CDR1, which contains V of Table 10 L any one of the CDR1 amino acid sequences having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity; (b) V L CDR2, which comprises V L any one of the CDR2 amino acid sequences having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity; and (c) V L CDR3, which comprises V L Any of the CDR3 amino acid sequences has an amino acid sequence with at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0314] In some embodiments, the ABP comprises: (a) a light chain variable domain (V H )CDR1, which comprises V H any one of the CDR1 amino acid sequences having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the ABP comprises (b) V HCDR2, which comprises V H Any of the CDR2 amino acid sequences has an amino acid sequence with at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity. In some embodiments, the ABP comprises V H CDR3, the V H CDR3 contains V corresponding to Table 10 H Any of the CDR3 amino acid sequences has an amino acid sequence with at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0315] In some embodiments, the ABP comprises: (a) a light chain variable domain (V H ) CDR1 comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 62-64. In some embodiments, the ABP comprises (b) V H CDR2 comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 65-69. In some embodiments, the ABP comprises V H CDR3, the V H The CDR3 comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 70-73.

[0316] In some embodiments, the ABP comprises: (a) V H CDR1, which comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SNTASWN (SEQ ID NO:62); SNSASWN (SEQ ID NO:63) and STTASWN (SEQ ID NO:64); (b) V H CDR2, which comprises an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 65-69; and (c) V HA CDR3 comprising an amino acid sequence having at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 70-73.

[0317] In some embodiments, the ABP comprises the same CDR sequences as an antibody selected from A1, C1, F1, G1, C2, F2, H2, H3, F12, and B8.

[0318] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 45. L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO: 65 H CDR2 and V having the sequence of SEQ ID NO: 70 H CDR3.

[0319] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 45. L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0320] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 46. L CDR1, V having the sequence of SEQ ID NO: 53 L CDR2, V having the sequence of SEQ ID NO: 56 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0321] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 47. L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 57 L CDR3, V having the sequence of SEQ ID NO: 64 H CDR1, V having the sequence of SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0322] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 48. L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 58 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3.

[0323] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 49. L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 67 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0324] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 49. L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 59 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 67 HCDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0325] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 50. L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 60 L CDR3, V having the sequence of SEQ ID NO: 62 H CDR1, V having the sequence of SEQ ID NO: 69 H CDR2 and V having the sequence of SEQ ID NO: 73 H CDR3.

[0326] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 47. L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3.

[0327] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 51. L CDR1, V having the sequence of SEQ ID NO: 54 L CDR2, V having the sequence of SEQ ID NO: 61 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 68 H CDR2 and V having the sequence of SEQ ID NO: 72 H CDR3.

[0328] In some embodiments, the ABP comprises a V having a sequence of SEQ ID NO: 45. L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 LCDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

[0329] In some embodiments, the ABP comprises an antibody. In some embodiments, the ABP is a monoclonal antibody. In some embodiments, the ABP is selected from a human antibody, a humanized antibody or a chimeric antibody. In some embodiments, the ABP is a single chain variable fragment (scFv). In some embodiments, the ABP comprises an antibody fragment. In some embodiments, the ABP comprises an immunoglobulin constant region.

[0330] In some embodiments, the ABP comprises a variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs:74-88 of Table 11.

[0331] Table 11: V of ABPs with Kabot position L and V H chain

[0332]

[0333]

[0334] In some embodiments, the ABP comprises a heavy chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 82-88 of Table 11.

[0335] In some embodiments, the ABP comprises a light chain variable domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or at least 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 74-81.

[0336] In some embodiments, the ABP comprises the heavy chain variable domain and the light chain variable domain of an antibody selected from A1, C1, F1, G1, C2, F2, H2, H3, F12, and B8.

[0337] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:82 and a light chain variable domain having the sequence of SEQ ID NO:74.

[0338] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:83 and a light chain variable domain having the sequence of SEQ ID NO:74.

[0339] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:84 and a light chain variable domain having the sequence of SEQ ID NO:75.

[0340] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:85 and a light chain variable domain having the sequence of SEQ ID NO:76.

[0341] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:86 and a light chain variable domain having the sequence of SEQ ID NO:77.

[0342] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:84 and a light chain variable domain having the sequence of SEQ ID NO:77.

[0343] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:84 and a light chain variable domain having the sequence of SEQ ID NO:78.

[0344] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:87 and a light chain variable domain having the sequence of SEQ ID NO:79.

[0345] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:88 and a light chain variable domain having the sequence of SEQ ID NO:80.

[0346] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:88 and a light chain variable domain having the sequence of SEQ ID NO:81.

[0347] In some embodiments, the ABP comprises a heavy chain variable domain having the sequence of SEQ ID NO:83 and a light chain variable domain having the sequence of SEQ ID NO:74.

[0348] In some embodiments, the ABP comprises a scFv. In some embodiments, the ABP is a scFv. In some embodiments, the scFv comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 89-98 of Table 12.

[0349] Table 12: scFv amino acid sequences of ABPs with Kabot positions

[0350]

[0351]

[0352]

[0353] In certain embodiments, the ABP comprises Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single-chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, a V domain antibody, a bispecific tandem bivalent scFv, or a bispecific T cell engager (BiTE).

[0354] In specific embodiments, the ABP comprises an Fc, optionally a human Fc.

[0355] In some embodiments, the ABP comprises a class of heavy chain constant regions selected from IgG, IgA, IgD, IgE, and IgM. In some embodiments, the ABP comprises a heavy chain constant region of the IgG1 class and a subclass selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the ABP comprises a heavy chain constant region of IgG. In certain embodiments, the ABP comprises a heavy chain constant region of IgG1.

[0356] In some embodiments, the ABP is bispecific or multispecific.In some embodiments, the ABP is afucosylated.

[0357] Also provided are kits comprising one or more pharmaceutical compositions comprising the ABP and instructions for use of the pharmaceutical compositions. Also provided are pharmaceutical compositions comprising the ABP described herein.

[0358] Also provided are isolated polynucleotides encoding the ABP or portions thereof provided herein. Also provided are vectors comprising such polynucleotides. Also provided are recombinant host cells comprising such polynucleotides and recombinant host cells comprising such vectors.

[0359] Also provided are methods of producing ABP using the polynucleotides, vectors or host cells provided herein. In some aspects, the disclosure provides a method of producing an ABP that specifically binds to human CD63, CD151, CD72, CD84, CD69 or CD109, comprising: expressing the ABP in a host cell and isolating the ABP.

[0360] 3.3.2 ABP-drug conjugates

[0361] Another aspect of the present disclosure provides antigen binding protein (ABP)-drug conjugates.

[0362] In some embodiments, the ABP-drug conjugate comprises an antigen binding protein (ABP), a cytotoxic agent linked to the ABP, and optionally a linker that links the cytotoxic agent to the ABP, wherein the ABP specifically binds a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109.

[0363] In some embodiments, the ABP-drug conjugate comprises one or more of the antigen binding proteins (ABPs) described in 3.3.1.

[0364] In certain embodiments, the ABP specifically binds CD63. In certain embodiments, the ABP specifically binds CD151. In certain embodiments, the ABP specifically binds CD72. In certain embodiments, the ABP specifically binds CD84. In certain embodiments, the ABP specifically binds CD69. In certain embodiments, the ABP specifically binds CD109.

[0365] In some embodiments, the ABP is human, humanized, or chimeric. In some embodiments, the ABP is monoclonal. In some embodiments, the ABP is bispecific or multispecific.

[0366] In some embodiments, the ABP comprises a Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V domain antibody.

[0367] In some embodiments, the ABP comprises an Fc, optionally a human Fc.

[0368] In some embodiments, the ABP comprises a class of heavy chain constant regions selected from IgG, IgA, IgD, IgE and IgM. In some embodiments, the ABP comprises a heavy chain constant region of the IgG class and a subclass selected from IgG1, IgG2, IgG3 and IgG4. In some embodiments, the ABP comprises a heavy chain constant region of IgG1. In some embodiments, the ABP comprises a variable heavy chain region. In some embodiments, the ABP comprises a variable light chain region. In some embodiments, the ABP comprises a variable heavy chain region and a variable light chain region. In some embodiments, the ABP is an antibody binding fragment. 3.4.1 "Antigen Binding Protein (ABP)" describes antibodies and antigen binding fragments.

[0369] In some embodiments, the cytotoxic agent comprises an anti-angiogenic agent, a pro-apoptotic agent, an anti-mitotic agent, an anti-kinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioisotope.

[0370] In some embodiments, the linker is a cleavable linker.In some embodiments, the linker is a non-cleavable linker.

[0371] In some embodiments, the main chain length of the joint is 100 atoms or less, such as 50 atoms or less or 20 atoms or less. In other embodiments, the main chain length of the joint is 100 atoms or longer. Joint or linker can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, such as 1 to 50 atoms in length or 1 to 20 atoms in length, such as about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbon atoms in length, wherein the joint can be straight chain, branched chain, cyclic or single atom. In certain embodiments, one, two, three, four or five or more carbon atoms of the joint main chain can be optionally replaced by sulfur, nitrogen or oxygen heteroatoms. The key between the main chain atoms can be saturated or undersaturated, and usually no more than one, two or three unsaturated bonds will be present in the joint main chain. Connecting base can include one or more substituents, such as substituents with alkyl, aryl or alkenyl. The linker may include, but is not limited to, oligo(ethylene glycol); ethers, thioethers, tertiary amines, alkyl groups, which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may include cyclic groups, such as aryl, heterocyclic or cycloalkyl groups, wherein 2 or more atoms, such as 2, 3 or 4 atoms of the cyclic group are included in the backbone. The linker may be peptidic or non-peptidic.

[0372] Non-limiting examples of antibody-drug conjugate linkers and chemistries can be found in US Patent Application Publication Nos.: US20200277306 and US20190328900A1; US ​​Patent No. US7750116B1, which are incorporated herein by reference in their entirety. Other non-limiting examples of ADC chemical compositions are described in Olivier et al., (2017) "Antibody-Drug Conjugates: Fundamentals, Drug Development, and Clinical Outcomes to Target Cancer" ISBN: 978-1-119-06068-0), which are incorporated herein by reference in their entirety.

[0373] Aspects of the present disclosure include polynucleotides encoding ABP-drug conjugates. In some embodiments, the polynucleotides further comprise sequences homologous to the target genomic region for site-specific integration. In some embodiments, polynucleotides are designed for gene editing using endonucleases such as CRISPR-cas systems, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and meganucleases.

[0374] In some embodiments, the polynucleotide is in a viral or non-viral vector. The vector can be used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0375] In some embodiments, the method comprises administering a non-viral vector comprising the polynucleotide or a pharmaceutical composition thereof. In some embodiments, a non-viral vector or non-viral method is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0376] Non-limiting examples of non-viral delivery methods that can be used in the methods of the present invention to deliver polynucleotides include, but are not limited to: physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical carriers, inorganic particles, calcium phosphate particles, magnetic particles, polymer carriers, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly (DL-lactide) (PLA) and poly (DL-lactide-co-glycoside) (PLGA), dendrimers or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06 and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are incorporated herein by reference in their entirety.

[0377] 3.3.3 Bispecific T cell engagers (BiTEs)

[0378] Aspects of the present disclosure include bispecific T cell engagers (BiTEs) comprising an antigen binding domain and a T cell activation domain.

[0379] In some embodiments, the antigen binding domain specifically binds a target antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD 109. In some embodiments, the antigen binding domain is any one of the ABPs described herein.

[0380] In certain embodiments, the antigen binding domain is an extracellular antigen binding domain. In certain embodiments, the antigen binding domain specifically binds CD63. In certain embodiments, the antigen binding domain specifically binds CD151. In certain embodiments, the antigen binding domain specifically binds CD72. In certain embodiments, the antigen binding domain specifically binds CD84. In certain embodiments, the antigen binding domain specifically binds CD69. In certain embodiments, the antigen binding domain specifically binds CD109.

[0381] An example of a BiTE is a fusion protein of an extracellular recognition domain (e.g., an antigen binding domain) and one or more T cell activation domains. Upon antigen binding, the intracellular signaling portion of the BiTE can trigger activation-related responses in T cell-specific molecules, thereby stimulating T cell activation, tumor killing, and / or cytokine production.

[0382] In some embodiments, the BiTE comprises an antigen binding domain that specifically binds a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109.

[0383] In some embodiments, a bispecific T cell engager (BiTE) may contain two scFvs produced as a single polypeptide chain. In certain embodiments, a BiTE comprises two ScFvs of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109). Methods for preparing and using BiTE antibodies are described in the art. See, e.g., Cioffi et al., Clin Cancer Res 18:465; Brischwein et al., Mol Immunol 43:1129-43 (2006); Amann M et al., Cancer Res 68:143-51 (2008); Schlereth et al., Cancer Res 65:2882-2889 (2005); and Schlereth et al., Cancer Immunol Immunother 55:785-796 (2006); Huehls A., et al., Immunol Cell Biol. 93(3):290-296 (2014); Wang et al., Antibodies. 8(32):1-30 (2019), which are incorporated herein by reference in their entireties.

[0384] In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109).

[0385] In some embodiments, the T cell activation domain comprises a single chain variable fragment (scFV) of an antibody that specifically binds to CD3. In some embodiments, the T cell activation domain comprises the intracellular domain of CD3ζ. In some embodiments, the T cell activation domain comprises a ZAP-70 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0386] In a non-limiting example, when both the antigen binding domain and the T cell activation domain comprise scFv, the scFv can be generated by linking the heavy chain and light chain of each Fv with a serine-glycine linker sequence.

[0387] In certain embodiments, the linker comprises one or more, two or more, three or more, four or more, five or more, or six or more repeats of GGGGS ("GGGGS" disclosed as SEQ ID NO: 2). In certain embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 2).

[0388] In certain embodiments, the linker comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more repeats of SGGGG ("SGGGG" disclosed as SEQ ID NO:3). In certain embodiments, the linker comprises the amino acid sequence SGGGG (SEQ ID NO:3).

[0389] In certain embodiments, the linker can make the peptide long enough and flexible to allow the heavy and light chains to associate in the normal conformation.

[0390] In some embodiments, the linker is a rigid linker, a cleavable linker, or a flexible linker.

[0391] In certain embodiments, the BiTE comprises a linker connecting the antigen binding domain and the T cell activation domain. As a non-limiting example, when both the antigen binding domain and the T cell activation domain comprise scFv, a GGGGS (SEQ ID NO: 2) repeat linker can connect the two scFvs.

[0392] In some embodiments, the linker comprises the amino acid sequence GGGGS (SEQ ID NO: 2). In certain embodiments, the linker comprises the amino acid sequence motif (G4S) n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 4). In some embodiments, the linker comprises the amino acid sequence SGGGG (SEQ ID NO: 3). In certain embodiments, the linker comprises the amino acid sequence motif (SG4) n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (SEQ ID NO: 5). In some embodiments, the linker connects two scFvs. In some embodiments, the linker comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more GGGGS repeats ("GGGGS" is disclosed as SEQ ID NO: 2).

[0393] In some embodiments, the joint comprises 1-25 amino acids. In some embodiments, the joint comprises 1-20 amino acids. In some embodiments, the joint comprises 1-15 amino acids. In certain embodiments, the joint comprises 1-10 amino acids. In certain embodiments, the joint comprises 1-9 amino acids. In certain embodiments, the joint comprises 1-8 amino acids. In certain embodiments, the joint comprises 1-7 amino acids. In some embodiments, the joint comprises 1-6 amino acids. In certain embodiments, the joint comprises 1-5 amino acids. In certain embodiments, the joint comprises 1-4 amino acids. In some embodiments, the joint comprises 1-2 amino acids. In certain embodiments, the joint comprises 1-10 amino acids. In certain embodiments, the length of the joint can determine the flexibility of movement between the two scFvs, and can be adjusted by including more or less joint repeats to optimize the combination with the two target cells. For example, in some embodiments, a short flexible joint connecting two scFvs can provide free rotation of the antigen binding domain and the T cell activation domain.

[0394] In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6).In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:7).

[0395] Non-limiting examples of linkers can be found in Chen et al., (Chen X., Zaro JL, Shen W.C. Fusion protein linkers: Property, design and functionality. Adv. Drug Deliv. Rev. 2013; 65: 1357-1369), which is incorporated herein by reference in its entirety.

[0396] In certain embodiments, the linker is selected from: (GGGGS)3 (SEQ ID NO: 8), (G)8 (SEQ ID NO: 9), (G)6 (SEQ ID NO: 10), (EAAAK)3 (SEQ ID NO: 11), (EAAAK) n (SEQ ID NO:12), where n is 1-3, A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:13), PAPAP (SEQ ID NO:14), AEAAAKEAAAKA (SEQ IDNO:15), (Ala-Pro) nwherein n is 5-17) (SEQ ID NO: 16), VSQTSKLTR↓AETVFPDV b (SEQ ID NO: 17), PLG↓LWA c (SEQ ID NO:18), RVL↓AEA (SEQ ID NO:19); EDVVCC↓SMSY (SEQ ID NO:20), GGIEGR↓GS c (SEQ ID NO:21), TRHRQPR↓GWE (SEQ ID NO:22), and AGNRVRR↓SVG (SEQ ID NO:23). a Protease-sensitive cleavage sites are indicated by “↓”; b Factor XIa / FVIIA-sensitive cleavage; c Matrix metalloproteinase-1 sensitive cleavage sequence, an example of which is provided here; d HIV PR (HIV-1 protease); NS3 protease (HCV protease); Factor Xa-sensitive cleavage; e Furin-sensitive cleavage; f Cathepsin B-sensitive cleavage.

[0397] Aspects of the disclosure include host cells comprising the bacteriophage.

[0398] In some embodiments, the antigen binding domain binds to an epitope on CD63, CD151, CD72, CD84, CD69, or CD109. In some embodiments, the antigen binding domain comprises the CDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. In some embodiments, the antigen binding domain comprises the V H and V L In some embodiments, the antigen binding domain comprises a CD63, CD151, CD72, CD84, CD69 or CD109 single chain variable fragment (scFv).

[0399] Aspects of the present disclosure include polynucleotides encoding BiTEs. In some embodiments, the polynucleotides also include sequences homologous to the target genomic region for site-specific integration. In some embodiments, polynucleotides are designed for gene editing using endonucleases such as CRISPR-cas systems, zinc finger nucleases, transcription activator-like effector nucleases (TALENs) and meganucleases.

[0400] Aspects of the present disclosure include vectors comprising polynucleotides. Any vector that can be used for gene delivery can be used. In some variants, viral vectors (such as AAV, adenovirus, lentivirus, retrovirus) are used. Non-limiting examples of vectors that can be used in the present disclosure include, but are not limited to, human immunodeficiency virus; HSV, herpes simplex virus; MMSV, Moloney murine sarcoma virus; MSCV, lentivirus, mouse stem cell virus; SFV, Semliki Forest virus; SIN, Sindbis virus; VEE, Venezuelan equine encephalitis virus; VSV, vesicular stomatitis virus; VV, and vaccinia virus.

[0401] In some embodiments, the vector is a recombinant AAV vector. In some embodiments, the vector used in the methods of the present disclosure is encapsulated into a viral particle (e.g., a viral particle includes but is not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16).

[0402] In some embodiments, the method comprises administering a vector comprising a polynucleotide encoding a BiTE or a pharmaceutical composition thereof. For example, a viral or non-viral vector can be used to modify T cells to promote specific targeting of original cells by expressing an exogenous BiTE. In some embodiments, a vector is used in vitro to generate immune response cells expressing a BiTE.

[0403] In some embodiments, the method comprises administering a vector comprising a polynucleotide encoding a BiTE or a pharmaceutical composition thereof.

[0404] In some embodiments, the method comprises administering a non-viral vector comprising the polynucleotide or a pharmaceutical composition thereof. In some embodiments, a non-viral vector or non-viral method is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0405] Non-limiting examples of non-viral delivery methods that can be used in the methods of the present invention to deliver polynucleotides include, but are not limited to: physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical carriers, inorganic particles, calcium phosphate particles, magnetic particles, polymer carriers, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly (DL-lactide) (PLA) and poly (DL-lactide-co-glycoside) (PLGA), dendrimers or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06 and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are incorporated herein by reference in their entirety.

[0406] 3.3.4 Chimeric Antigen Receptor (CAR)

[0407] Aspects of the present disclosure include chimeric antigen receptors (CARs) comprising an extracellular antigen binding domain, a transmembrane domain, a signaling domain, and optionally a co-stimulatory domain.

[0408] In some embodiments, the extracellular antigen binding domain specifically binds to a target antigen selected from CD63, CD151, CD72, CD84, CD69, and CD109. In certain embodiments, the antigen binding domain specifically binds CD63. In certain embodiments, the antigen binding domain specifically binds CD151. In certain embodiments, the antigen binding domain specifically binds CD72. In certain embodiments, the antigen binding domain specifically binds CD84. In certain embodiments, the antigen binding domain specifically binds CD69. In certain embodiments, the antigen binding domain specifically binds CD109.

[0409] In some embodiments, the antigen binding domain is any of the ABPs described herein.

[0410] The example of CAR is a fusion of an extracellular recognition domain (e.g., an antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains. After antigen binding, the intracellular signaling portion of CAR triggers activation-related responses in immune cells, such as releasing cytolytic molecules to induce tumor cell death, etc.

[0411] In some embodiments, CAR comprises an extracellular antigen binding domain, a transmembrane domain, a signaling domain, and an optional co-stimulatory domain, wherein the extracellular antigen binding domain specifically binds to a target protein / antigen selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109. In certain embodiments, CAR also comprises a hinge region of a polypeptide.

[0412] In some embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv) of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69, or CD109).

[0413] In some embodiments, the signaling domain comprises the intracellular domain of CD3ζ. In some embodiments, the signaling domain comprises the ZAP-70 intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0414] In some embodiments, one or more (for example, two or more, three or more, four or more or five or more) costimulatory domains are also included, wherein the costimulatory domain is CD28 costimulatory domain, 4-1BB costimulatory domain, CD27 costimulatory domain, OX40 costimulatory domain or ICOS costimulatory domain.In some embodiments, costimulatory domain is selected from 4-1BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR and HVEM.

[0415] In some embodiments, CAR comprises 4-1BB, CD28 or a fragment thereof. In certain embodiments, CAR comprises a CD28 transmembrane domain and 4-1BB. In certain embodiments, CAR comprises a hinge region, a CD28 transmembrane domain and 4-1BB. In certain embodiments, the hinge region is from a CD28 polypeptide.

[0416] In some embodiments, the CAR comprises a hinge region of a CD28 polypeptide, wherein the hinge region comprises at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the following amino acid sequence: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 99).

[0417] In some embodiments, the CAR comprises a transmembrane domain of a CD28 polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the following amino acid sequence: FWV L VVVGGV L ACYSLLVTVAFIIFWV (SEQ ID NO: 100).

[0418] In some embodiments, the CD28 costimulatory domain has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the following amino acid sequence: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV L VVV GGV L ACYSLLVTVAFIIFWV (SEQ ID NO: 102).

[0419] In some embodiments, the zeta (CD3 zeta) signaling domain is RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV L The amino acid sequence of DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 101) has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

[0420] In some embodiments, the CAR comprises a CD28 transmembrane (TM) and a 4-1BB co-stimulatory domain combined with a zeta (CD3zeta) signaling domain.

[0421] In some embodiments, the CAR construct is placed in a plasmid, such as a pUC57 plasmid. Using restriction endonuclease technology, the coding sequence of the CAR can be cut and placed in a vector transfer plasmid for high titer viral vector production (see, e.g., Fig.13 and 14 ).

[0422] In some embodiments, CAR comprises a signal peptide (e.g., a signal interfering peptide (SIP)). In certain embodiments, the signal peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the following amino acid sequence: MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 103). In certain embodiments, the signal peptide is encoded by a nucleic acid sequence comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the following nucleotide sequence: ATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGCAGCTCCCAG ATGGGTCCTGTCC (SEQ ID NO: 104).

[0423] In some embodiments, the extracellular antigen binding domain is a single chain variable fragment (scFv). In some embodiments, the antigen binding domain comprises an scFv of an antibody that specifically binds to a target protein (CD63, CD151, CD72, CD84, CD69 and / or CD109). In certain embodiments, the scFv binds to the target protein CD63. In certain embodiments, the scFv binds to the target protein CD151. In certain embodiments, the scFv binds to the target protein CD72. In certain embodiments, the scFv binds to the target protein CD84. In certain embodiments, the scFv binds to the target protein CD69. In certain embodiments, the scFv binds to the target protein CD109. In some embodiments, the scFv comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 89-98 of Table 12.

[0424] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:89.

[0425] In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv, as above, having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO:89.

[0426] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 90. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 90.

[0427] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 91. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 91.

[0428] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 92. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 92.

[0429] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 93.

[0430] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 94.

[0431] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0432] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 96.

[0433] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 97.

[0434] In some embodiments, the scFv comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments, the scFv comprises a nucleotide sequence encoding a scFv region, wherein the nucleotide sequence encodes a scFv having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 98.

[0435] In some embodiments, the CAR comprises a signal peptide, a scFv region, one or more co-stimulatory domains, and a signaling domain.

[0436] Table 14 provides the nucleotide sequences of the CAR constructs (SEQ ID NOs: 24-34), and Table 14 provides the protein sequences of the CAR constructs (SEQ ID NOs: 35-44 and 36), respectively, in the order of appearance.

[0437] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 35-44 of Table 13 (as provided in Table 13).

[0438] Table 13: CAR construct protein sequences

[0439]

[0440]

[0441]

[0442]

[0443] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 24 of Table 14.

[0444] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 25 of Table 14.

[0445] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 37 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 26 of Table 14.

[0446] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 38 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 27 of Table 14.

[0447] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 39 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 28 of Table 14.

[0448] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 29 of Table 14.

[0449] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 41 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 30 of Table 14.

[0450] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 42 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 31 of Table 14.

[0451] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 43 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 32 of Table 14.

[0452] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 44 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 33 of Table 14.

[0453] In some embodiments, the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 36 of Figure 27. In some embodiments, the CAR comprises a nucleic acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 34 of Table 14.

[0454] Table 14: CAR construct cDNA sequences

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464] Aspects of the present disclosure include host cells comprising a CAR. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a CD4 + and CD8 + In certain embodiments, the host cell is a natural memory cell (T n + ) T cells. In some embodiments, the host cell is a stem cell memory (T scm) T cells. In some embodiments, the host cell is a stem cell memory (T cm , CD197[CCR7] + CD45R0 + ) T cells. In some embodiments, the host cell is a central memory (T cm , CD197(CCR7) + CD45R0 + ) T cells. CD197 is used interchangeably herein as "CCR7".

[0465] Aspects of the present disclosure include one or more host cells comprising a CAR. In certain embodiments, the one or more host cells comprising the CAR are selected from natural and stem cell memory (T n + T scm , CD197[CCR7 + ] + CD45R0-) and central memory (T cm , CD197(CCR7) + CD45R0 + )T cells. In some embodiments, the one or more cells comprising the CAR are natural killer (NK) cells.

[0466] In some embodiments, CAR comprises: an extracellular antigen binding domain, a transmembrane domain, and a signaling domain. In some embodiments, CAR is a second generation CAR, which comprises an extracellular antigen binding domain, a transmembrane domain, a signaling domain, and a co-stimulatory domain. In some embodiments, CAR comprises: an extracellular antigen binding domain, a hinge region of a polypeptide, a transmembrane domain, and a signaling domain.

[0467] In some embodiments, the extracellular antigen binding domain binds to an epitope on a CD63, CD151, CD72, CD84, CD69, or CD109 target antigen. In some embodiments, the extracellular antigen binding domain comprises the CDRs of a CD63, CD151, CD72, CD84, CD69, or CD109 antibody. In some embodiments, the extracellular antigen binding domain comprises the V H and V L In some embodiments, the extracellular antigen binding domain comprises a CD63, CD151, CD72, CD84, CD69 or CD109 single chain variable fragment (scFv).

[0468] Aspects of the present disclosure include polynucleotides encoding CAR. In some embodiments, the polynucleotides also include sequences homologous to the target genomic region for site-specific integration. In some embodiments, polynucleotides are designed for gene editing using endonucleases such as CRISPR-cas systems, zinc finger nucleases, transcription activator-like effector nucleases (TALENs) and meganucleases.

[0469] Aspects of the present disclosure include vectors comprising polynucleotides. Any vector that can be used for gene delivery can be used. In some variants, viral vectors (such as AAV, adenovirus, lentivirus, retrovirus) are used. Non-limiting examples of vectors that can be used in the present disclosure include, but are not limited to, human immunodeficiency virus; HSV, herpes simplex virus; MMSV, Moloney murine sarcoma virus; MSCV, lentivirus, mouse stem cell virus; SFV, Semliki Forest virus; SIN, Sindbis virus; VEE, Venezuelan equine encephalitis virus; VSV, vesicular stomatitis virus; VV, and vaccinia virus.

[0470] In some embodiments, the vector is a lentiviral vector. Lentiviruses are a subclass of retroviruses. However, lentiviruses can integrate into the genome of non-dividing cells, while retroviruses can only infect dividing cells.

[0471] Lentiviral vectors are usually produced by a packaging cell line, usually HEK293, transformed with several plasmids. The plasmids include (1) a packaging plasmid encoding virion proteins such as capsid and reverse transcriptase, and (2) a plasmid containing the foreign gene to be delivered to the target.

[0472] When the virus enters the cell, the viral genome in RNA form is reverse transcribed to produce DNA, which is then inserted into the genome by the viral integrase. Therefore, the foreign substance delivered with the lentiviral vector can be retained in the genome and passed on to the cell's progeny when it divides.

[0473] In some embodiments, the vector is a recombinant AAV vector. In some embodiments, the vector used in the methods of the present disclosure is encapsulated into a viral particle (e.g., an AAV viral particle includes but is not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16).

[0474] Adeno-associated viruses are able to infect non-dividing cells and various cell types, making them useful in constructing the gene delivery systems of the present disclosure. Detailed descriptions of the use and preparation of AAV vectors are provided in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0475] The results of research on AAV as a gene delivery system are disclosed in LaFace et al, Viology, 162: 483486 (1988); Zhou et al., Exp. Hematol. (NY), 21: 928-933 (1993); Walsh et al, J. Clin. Invest., 94: 1440-1448 (1994) and Flotte et al., Gene Therapy, 2: 29-37 (1995). Generally, recombinant AAV viruses are prepared by co-transfection of a plasmid containing a target gene (i.e., a decorin gene and a target nucleotide sequence to be delivered) flanked by two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989) and an expression plasmid containing a wild-type AAV coding sequence and no terminal repeats (McCarty et al., J. Viral., 65: 2936-2945 (1991)).

[0476] In some embodiments, the method includes administering a vector or a pharmaceutical composition thereof comprising a polynucleotide encoding CAR. For example, a viral or non-viral vector can be used to modify T cells to promote the specific targeting of primitive cells by the expression of exogenous CAR. In some embodiments, the vector is used to produce immune response cells (e.g., CAR-T cells) expressing CAR in vitro. Aspects of the present disclosure include immune response cells expressing CAR. Aspects of the present disclosure include immune response cells comprising polynucleotides encoding CAR or vectors comprising polynucleotides.

[0477] In some embodiments, the method comprises administering a non-viral vector comprising the polynucleotide or a pharmaceutical composition thereof. In some embodiments, a non-viral vector or non-viral method is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0478] Non-limiting examples of non-viral delivery methods that can be used in the methods of the present invention to deliver polynucleotides include, but are not limited to: physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical carriers, inorganic particles, calcium phosphate particles, magnetic particles, polymer carriers, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly (DL-lactide) (PLA) and poly (DL-lactide-co-glycoside) (PLGA), dendrimers or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06 and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are incorporated herein by reference in their entirety.

[0479] In some embodiments, the immune response cell is a bispecific CAR-T targeting two target proteins selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the immune response cell is a bispecific CAR-T targeting (i) a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109; and (ii) CD3. In some embodiments, the immune response cell is a bispecific CAR-T targeting (i) a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109; and (ii) CD123.

[0480] In some embodiments, the immune response cell is an αβT cell, a γδT cell, or a natural killer (NK) cell. In some embodiments, the αβT cell is a CD3 + T cells or CD4 + T cells or CD8 + T cells.

[0481] One aspect of the present disclosure includes a method for preparing immune response cells. In some embodiments, the method includes transfecting or transducing a polynucleotide encoding a CAR or a vector comprising a polynucleotide encoding a CAR into an immune response cell. In some embodiments, the method includes amplifying the immune response cell for at least 48 hours. In some embodiments, the immune response cell is transduced with a multiplicity of infection of at least 10.

[0482] In some embodiments, the method further comprises, after transduction, washing the vector, and expanding the CAR T cells for at least 14 days.

[0483] Transduction efficiency can be measured by digital droplet PCR (ddPCR) and can be expressed as vector copy number (VCN) / cell. In some embodiments, the average VCN of immune responsive CAR-T cells ranges from 2.5 to 33.5 (e.g., 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 17.5, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 29, 29.5, 30, 30.5, 31.5, 32, 32.5, 33, 33.5, etc.).

[0484] In some embodiments, after the polynucleotides encoding CAR or the vectors comprising the polynucleotides encoding CAR are transduced into immune response cells, the method includes measuring the expansion rate of immune response cells. In certain embodiments, the expansion rate of immune response cells is 2-20 times (e.g., 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 times).

[0485] In some embodiments, the immune response cells are naive and stem cell memory (Tn+T scm , CD197[CCR7] + CD45R0-) and / or central memory (T cm , CD197(CCR7) + CD45R0 + )T cells.

[0486] In some embodiments, immune response CAR T cells show the same degree of activation and exhaustion as empty CAR-T cells. This can be shown by upregulation of CD25, CD154, CD107a and CD137, as well as CD366, CD223 and CD279. In certain embodiments, immune response cells containing CAR do not change T cell function.

[0487] 3.4 Diagnostic methods

[0488] In one aspect, the present disclosure provides a method for diagnosing cancer. In particular, the method can be used to diagnose a bone marrow disorder or acute leukemia in a subject. The method includes the step of detecting the presence or absence or level of a tumor-specific antigen in a biological sample of the subject, wherein the tumor-specific antigen is selected from the group consisting of: CD63, CD151, CD72, CD84, CD69, and CD109.

[0489] In some embodiments, the detecting step comprises contacting the biological sample with an ABP, wherein the ABP specifically binds to a tumor-specific antigen. In some embodiments, the ABP is an antibody or antigen-binding fragment that binds to a tumor-specific antigen.

[0490] ABPs that can be used in the detection step are described in 3.3.1 "Antigen Binding Proteins (ABPs)".

[0491] In some embodiments, the detecting step comprises flow cytometry, immunocytochemistry, immunohistochemistry, fluorescence, or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the ABP is labeled. In some embodiments, the ABP is labeled with a fluorophore or an enzyme.

[0492] In some embodiments, the detecting step comprises measuring the mRNA level of the tumor-specific antigen in the biological sample. In some embodiments, the mRNA level is measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR) or by next generation sequencing.

[0493] In some embodiments, the method further comprises the step of performing a treatment based on the diagnosis.

[0494] 3.4.1 Biological samples

[0495] In some embodiments, the biological sample is a blood sample of the subject. In certain embodiments, the blood sample is a peripheral blood sample. In some embodiments, the biological sample is a bone marrow sample. In some embodiments, the biological sample comprises a solid tumor or a liquid tumor. In some embodiments, the biological sample is a xenograft (PDX) derived from an AML patient.

[0496] In some embodiments, the biological sample comprises primitive cells. In certain embodiments, the primitive cells are selected from bone marrow primitive cells (e.g., myeloblasts), lymphoid primitive cells, or a combination of bone marrow and lymphoid primitive cells. In some embodiments, the primitive cells are bone marrow primitive cells. In some embodiments, the primitive cells are lymphoid primitive cells. In some embodiments, the primitive cells are a combination of bone marrow and lymphoid primitive cells. In some embodiments, the biological sample is an AML primitive cell.

[0497] 3.4.2 Detection of the presence or level of TSA

[0498] In some embodiments, the method includes detecting the presence / absence or level (amount) of one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method includes detecting the presence / absence or level of two TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method includes detecting the presence / absence or level of three TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method includes detecting the presence / absence or level of four TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method includes detecting the presence / absence or level of five TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109. In some embodiments, the method comprises detecting the presence / absence or levels of six TSAs selected from CD63, CD151, CD72, CD84, CD69, and CD109.

[0499] In some embodiments, the detecting step comprises contacting the biological sample with an ABP. In some embodiments, the ABP specifically binds to one of the tumor-specific antigens. In some embodiments, the ABP is an anti-CD63 antibody. In some embodiments, the ABP is an anti-CD151 antibody. In some embodiments, the ABP is an anti-CD72 antibody. In some embodiments, the ABP is an anti-CD84 antibody. In some embodiments, the ABP is an anti-CD69 antibody. In some embodiments, the ABP is an anti-CD109 antibody.

[0500] In some embodiments, the ABP is labeled. In some embodiments, the ABP is labeled with a fluorophore or an enzyme. In some embodiments, the ABP is labeled with a radioisotope. In some embodiments, the ABP is coupled to alkaline phosphatase, horseradish peroxidase, beta-galactosidase, tobacco etch virus nucleo-endosomal-alpha endopeptidase ("TEV protease"). In some embodiments, the ABP is conjugated to a fluorophore selected from the group consisting of 1,8-ANS, 4-methylumbelliferone, 7-amino-4-methylcoumarin, 7-hydroxy-4-methylcoumarin, acridine, Alexa Fluor 350.TM., Alexa Fluor 405.TM., AMCA, AMCA-X, ATTO Rho6G, ATTO Rho11, ATTO Rho12, ATTO Rho13, ATTO Rho14, ATTO Rho101, Pacific Blue, Alexa Fluor 43Q.TM.Alexa Fluor480.TM., Alexa Fluor488.TM., BODIPY 492 / 515, AlexaFluor 532.TM., Alexa Fluor 546.TM., Alexa Fluor555.TM.Alexa Fluor594.TM.BODIPY505 / 515, Cy2, cyQUANT GR, FITC, Fluo-3, Fluo-4, GFP (EGFP), mHoneydew, OregonGreen.TM.488, Oregon Green.TM.514, EYFP, DsRed, DsRed2, dTomato, Cy3.5, Phycoerythrin (PE), Rhodamine Red, mTangerine, mStrawberry, mOrange, mBanana, Tetramethylrhodamine (TRITC), R-phycoerythrin, ROX, DyLight 594, Calcium Deep Red, Alexa Fluor594.TM., Alexa Fluor610.TM., TexasRed, mCherry, mKate, Alexa Fluor660.TM., Alexa Fluor680.TM., allophycocyanin, DRAQ-5, carboxy naphthofluorescein, C7, DyLight 750, Cellvue NIR780, DM-NERF, eosin, erythrosine, fluorescein, FAM, hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue, methoxycoumarin, naphthofluorescein, PyMPO, 5-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, 5-carboxy-2',4',5',7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylamino, Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, dansyl chloride, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresol fast violet, cresol blue violet, brilliant cresol blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptates, europium terpyridine diamine, europium cryptates or chelates, diamines, biscyanines, and La Jolla blue dye. In some embodiments, the ABP is conjugated to quantum dots. .

[0501] In some embodiments, the detecting step comprises flow cytometry, immunohistochemistry, immunofluorescence, or enzyme-linked immunosorbent assay (ELISA). In some embodiments, the detecting step comprises Western blot. In some embodiments, the detecting step comprises mass spectrometry.

[0502] In some embodiments, the detecting step comprises measuring the mRNA level of a tumor-specific antigen in a biological sample. In certain embodiments, the method comprises measuring the mRNA level of CD63. In certain embodiments, the method comprises measuring the mRNA level of CD151. In certain embodiments, the method comprises measuring the mRNA level of CD72. In certain embodiments, the method comprises measuring the mRNA level of CD84. In certain embodiments, the method comprises measuring the mRNA level of CD69. In certain embodiments, the method comprises measuring the mRNA level of CD109.

[0503] In some embodiments, mRNA levels are measured by in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR), or by sequencing (eg, next generation sequencing).

[0504] In some embodiments, the method further comprises the step of determining the presence or absence of cancer in the subject based on the presence / absence or level of TSA in a sample from the subject.

[0505] In some embodiments, when the subject shows expression of at least one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, when the subject shows expression of at least two, three, four or five TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL).

[0506] In some embodiments, when the subject shows expression of one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, when the subject shows expression of two, three, four or five TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, when the subject shows expression of CD63, CD151, CD72, CD84, CD69 and CD109, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL).

[0507] In some embodiments, when the subject shows an increased expression of at least one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109 compared to the control sample, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, when the subject shows an increased expression of one TSA selected from CD63, CD151, CD72, CD84, CD69 and CD109 compared to the control sample, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, when the subject shows an increased expression of two, three, four or five TSAs selected from CD63, CD151, CD72, CD84, CD69 and CD109 compared to the control sample, it is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, when a subject shows increased expression of CD63, CD151, CD72, CD84, CD69, and CD109 compared to a control sample, the subject is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL).

[0508] In some embodiments, a subject is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL) when the subject exhibits increased expression of at least one TSA selected from CD63, CD151, CD72, CD84, CD69, CD109 and further selected from CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3, and HLA-DR as compared to a control sample. In some embodiments, a subject is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL) when the subject exhibits increased expression of one TSA selected from CD63, CD151, CD72, CD84, CD69, CD109 and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine TSAs further selected from CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3, and HLA-DR as compared to a control sample. In some embodiments, a subject is diagnosed as having a myeloid disorder (MD) or acute leukemia (AL) when the subject exhibits increased expression of two, three, four or five TSAs selected from CD63, CD151, CD72, CD84, CD69, CD109 and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight or at least nine TSAs further selected from CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3 and HLA-DR as compared to a control sample. In some embodiments, a subject is diagnosed with a myeloid disorder (MD) or acute leukemia (AL) when the subject exhibits increased expression of CD63, CD151, CD72, CD84, CD69, CD109, and at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine additional TSAs selected from CD19, CD34, CD33, CD7, CD38, CD117, CD45, CD3, and HLA-DR as compared to a control sample.

[0509] In one embodiment, the subject is diagnosed with MD or AL when the subject shows an increase in expression of TSA of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to a predetermined reference level of TSA or compared to a sample from a healthy subject. In some embodiments, the subject is diagnosed with MD or AL when the subject shows an increase in expression of TSA of at least 2, 3, 4, 5, 10, 20, 50, or 100-fold compared to a predetermined reference level of TSA or compared to a sample from a healthy subject.

[0510] In some embodiments, the method further comprises the step of determining the presence or absence of cancer in the subject based on detecting the presence or level of the tumor-specific antigen in a sample from the subject.

[0511] 3.4.3. Bone marrow disorders and acute leukemia

[0512] In some embodiments, the bone marrow disorder is a bone marrow malignancy. In some embodiments, the bone marrow disorder is a myeloid leukemia. In certain embodiments, the myeloid leukemia is acute myeloid leukemia (AML). In certain embodiments, the myeloid leukemia is childhood acute myeloid leukemia.

[0513] In some embodiments, the bone marrow disorder is a bone marrow tumor. In some embodiments, the bone marrow disorder is selected from myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), myelodysplastic / myeloproliferative neoplasms (MDS / MPN), and myeloid malignancies associated with eosinophilia and platelet or fibroblast growth factor receptor abnormalities.

[0514] In some embodiments, the MDS is selected from refractory pancytopenia with monolineage dysplasia (refractory anemia; refractory neutropenia, refractory thrombocytopenia), refractory cyclic polycythemia, refractory pancytopenia with multilineage dysplasia, refractory polycythemia-1, refractory polycythemia-2, myelodysplastic syndrome with isolated del(5q), and myelodysplastic syndrome. In some embodiments, the MPN is selected from chronic myeloid leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic neutrophilic leukemia, chronic eosinophilic leukemia, hypereosinophilic syndrome, and mast cell disease. In some embodiments, the MDS / MPN is selected from chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, and atypical chronic myeloid leukemia. In some embodiments, the myeloid neoplasm is selected from a myeloid neoplasm associated with PDGFRA rearrangement, a myeloid neoplasm associated with PDGFRB rearrangement, and a myeloid neoplasm associated with FGFR1 rearrangement (such as 8p11 myeloproliferative syndrome). In some embodiments, the acute leukemia is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), lymphocytic leukemia (LL), myeloid leukemia (ML).

[0515] In some embodiments, bone marrow disorders and acute leukemias strike in children or adults. In some embodiments, bone marrow disorders and acute leukemias are childhood AML. In certain embodiments, bone marrow disorders and acute leukemias strike in subjects aged 1 to 18 years. In certain embodiments, bone marrow disorders and acute leukemias strike in subjects aged 1 day to 18 years. In certain embodiments, bone marrow disorders and acute leukemias strike in subjects aged 1 day to 1 year.

[0516] In some embodiments, the bone marrow disorders and acute leukemias are adult AML. In certain embodiments, the bone marrow disorders and acute leukemias occur in adults older than 18 years, older than 20 years, older than 25 years, older than 30 years, older than 35 years, older than 40 years, older than 45 years, older than 50 years, older than 55 years, older than 60 years, or older than 65 years.

[0517] 3.5 Treatment methods

[0518] In another aspect, the present disclosure provides a method of treating a subject with a hematological malignancy. In some embodiments, the subject has a refractory disease. In some embodiments, the subject has a relapsed disease.

[0519] In certain embodiments, the hematological malignancy is a refractory B-cell malignancy. B-cell malignancies include, but are not limited to, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia / small lymphocytic lymphoma, monoclonal B-cell lymphocytosis, B-cell prolymphocytic leukemia, splenic marginal zone lymphoma, hairy cell leukemia, splenic B-cell lymphoma / leukemia, unclassifiable, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia variant, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, monoclonal gammopathy of undetermined significance (MGUS) IgM, m heavy chain disease, g heavy chain disease, alpha heavy chain disease, MGUS IgG / A, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, monoclonal immunoglobulin deposition disease, extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma), nodal marginal zone lymphoma, nodal marginal zone lymphoma of pediatric origin, follicular lymphoma, follicular neoplasm in situ, duodenal follicular lymphoma, pediatric follicular lymphoma, IRF4-rearranged large B-cell lymphoma, primary cutaneous follicle center lymphoma, mantle cell lymphoma, mantle cell tumor in situ, diffuse large B-cell lymphoma (DLBCL) NOS, including germ center B-cell type and activated B-cell type; T-cell / histiocyte-rich large B-cell lymphoma, primary DLBCL of the central nervous system, primary cutaneous DLBCL, leg type, EBV+DLBCL NOS, EBV+ mucosal ulceration, DLBCL associated with chronic inflammation, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, plasmacytoid lymphoma, primary effusion lymphoma, HHV8+ DLBCL NOS, Burkitt lymphoma, Burkitt-like lymphoma with llq aberration, high-grade B-cell lymphoma with rearrangements of MYC and BCL2 and / or BCL6, high-grade B-cell lymphoma NOS, and B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and conventional Hodgkin lymphoma. In some embodiments, the malignant tumor treated with the anti-CD72 nanobody described herein is Hodgkin's lymphoma, such as Hodgkin's lymphoma with a predominance of nodular lymphocytes or classical Hodgkin's lymphoma, including nodular sclerosis typical Hodgkin's lymphoma, lymphocyte-rich typical Hodgkin's lymphoma, mixed cell typical Hodgkin's lymphoma and lymphocyte-depleted typical Hodgkin's lymphoma. In some embodiments, the subject suffers from post-transplant lymphoproliferative disease (PTLD), such as plasma cell hyperplasia PTLD, infectious mononucleosis PTLD, flocculent follicular hyperplasia PTLD, polymorphic PTLD, monomorphic PTLD (B- and T- / NK- cell type) or typical Hodgkin's lymphoma PTLD.

[0520] In one aspect, the present disclosure provides a method for treating a subject with a hematological malignancy, such as a malignant B cell or a malignant tumor including a malignant myeloid cell. In some embodiments, the hematological malignancy is a B cell leukemia. In some embodiments, the B cell leukemia is a chronic lymphocytic leukemia. In certain embodiments, the B cell leukemia is a mixed lineage leukemia (MLL). In certain embodiments, the hematological malignancy is a non-Hodgkin lymphoma. In certain embodiments, the hematological malignancy is a multiple myeloma. In some embodiments, the hematological malignancy comprises bone marrow cells expressing any one of the target proteins selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109. In some embodiments, the hematological malignancy comprises one or more, two or more, three or more, four or more, or five or more target proteins.

[0521] In one aspect, the present disclosure provides methods of treating a subject having a myeloid disorder (MD) or acute leukemia (AL). In some embodiments, the acute leukemia is acute lymphoblastic leukemia.

[0522] In some embodiments, the subject suffers from myeloblastic (M0) type AML. In some embodiments, the subject suffers from myeloblastic (M1) type AML. In some embodiments, the subject suffers from myeloblastic (M2) type AML. In some embodiments, the subject suffers from promyelocytic (M3) type AML. In some embodiments, the subject suffers from myelomonocytic (M4) type AML. In some embodiments, the subject suffers from mononuclear (M5) type AML. In some embodiments, the subject suffers from erythroleukemia (M6) type AML or AML type AML. In some embodiments, the subject suffers from megakaryocyte (M7) type AML or AML-AML.

[0523] In some embodiments, the subject comprises one or more, two or more, three or more, four or more, or five or more of the target protein.

[0524] In some embodiments, the method of the present disclosure includes the step of administering to a subject an effective amount of a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109. The therapeutic agent may be an antigen binding protein (ABP) as described herein, an ABP-drug conjugate, a bispecific T cell adapter (BiTE), or an immune response cell expressing a chimeric antigen receptor (CAR) specific for a target protein.

[0525] In certain embodiments, the therapeutic agent is a CAR-T cell. In certain embodiments, the therapeutic agent is a CAR-NK cell. In certain embodiments, CAR-T cells or CAR-NK cells do not cause hematological toxicity after being administered to a subject. In some embodiments, the method for treating with CAR-T cells or CAR-NK cells provided herein does not require supportive therapy. In some embodiments, the method includes administering a therapeutically effective amount of immune response cells comprising CAR, and the immunoglobulin therapy or autologous / allogeneic hematopoietic stem cell therapy (HSC) for rescuing hematopoiesis is not performed after the administration step, or is not combined with immunoglobulin therapy or autologous / allogeneic hematopoietic stem cell therapy (HSC) for rescuing hematopoiesis. In some embodiments, the immunoglobulin therapy is intravenous immunoglobulin (IVIG) treatment.

[0526] In some embodiments, the ABP or pharmaceutical composition is not administered in combination with an immunotherapeutic agent. In some embodiments, the ABP or pharmaceutical composition is not administered with a combination therapy. In some embodiments, the ABP or pharmaceutical composition is not administered with immunotherapy. In some embodiments, the method includes administering a therapeutically effective amount of an immune response cell comprising a CAR, and the administration step is followed by no autologous / allogeneic hematopoietic stem cell therapy for hematopoiesis rescue, or is not combined with autologous / allogeneic hematopoietic stem cell therapy for hematopoiesis rescue.

[0527] In some embodiments, the method further comprises the step of treating the subject with a chemotherapeutic agent or hematopoietic stem cell prior to administering the ABP, pharmaceutical composition, or immune response cell. In some embodiments, the subject has a refractory disease. In some embodiments, the subject has a refractory cancer. In some embodiments, the subject has a relapsed disease. In some embodiments, the subject is unresponsive to treatment with a chemotherapeutic agent or hematopoietic stem cell transplant.

[0528] In some embodiments, the therapeutic agent is administered in an amount sufficient to affect the survival of cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In certain embodiments, the therapeutic agent is administered in an amount sufficient to eliminate cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to reduce or kill cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to reduce or kill cancer cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to inhibit the biological effects associated with CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, the therapeutic agent is administered in an amount sufficient to affect the survival of cancer cells in a subject.

[0529] In some embodiments, the method of the present disclosure includes the step of administering to the subject an effective amount of a therapeutic agent that specifically binds to a target protein selected from the group consisting of: CD63, CD151, CD72, CD84, CD69, and CD109, wherein the treatment is the first treatment (e.g., the first therapy or the first therapy line) administered to the subject for the treatment of a hematological malignancy. In some embodiments, the method of the present disclosure includes the step of administering to the subject an effective amount of a therapeutic agent that specifically binds to a target protein selected from the group consisting of: CD63, CD151, CD72, CD84, CD69, and CD109, wherein the treatment is the second treatment (e.g., the second therapy or the second therapy line) administered to the subject for the treatment of a hematological malignancy. For example, in certain embodiments, prior to treatment with a therapeutic agent that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109, the subject has undergone a first therapy for the treatment of a hematological malignancy. In certain embodiments, the first therapy is selected from one or more of the following groups: chemotherapy and hematopoietic stem cell transplantation therapy.

[0530] In some embodiments, the therapeutic agent for a target protein specifically binding to a subject selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109 is a first-line therapy. In some embodiments, the therapeutic agent for a target protein specifically binding to a subject selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109 is a second-line therapy. In certain embodiments, the therapeutic agent for a target protein specifically binding to a subject selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109 is a first-line therapy. In certain embodiments, the therapeutic agent for a target protein specifically binding to a subject selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109 is not combined with any other cancer therapy.

[0531] In some embodiments, before treatment with a therapeutic agent for a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, the subject suffers from refractory disease. In certain embodiments, before treatment with a therapeutic agent for a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, the subject has stopped responding to cancer treatment. In certain embodiments, the subject is a recurrent subject, wherein before treatment with a therapeutic agent for a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, cancer cells are present in the patient after cancer treatment. In certain embodiments, before treatment with a therapeutic agent for a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, the subject does not respond to first-line therapy. In certain embodiments, the patient was unresponsive to a first line of therapy prior to treatment with a therapeutic agent that specifically binds a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69, and CD109.

[0532] In some embodiments, the subject is an adult. In certain embodiments, the subject is an adult who is older than 18 years old, older than 20 years old, older than 25 years old, older than 30 years old, older than 35 years old, older than 40 years old, older than 45 years old, older than 50 years old, older than 55 years old, older than 60 years old, or older than 65 years old. In certain embodiments, the subject is a child subject. In certain embodiments, the subject is less than 18 years old, less than 20 years old, less than 25 years old, less than 30 years old, less than 35 years old, less than 40 years old, less than 45 years old, less than 50 years old, less than 55 years old, less than 60 years old, or less than 65 years old.

[0533] Aspects of the present disclosure include a polynucleotide encoding an ABP, an ABP-drug conjugate, a bispecific T cell adapter (BiTE) or a chimeric antigen receptor (CAR). In some embodiments, the polynucleotide also includes a sequence homologous to the target genomic region for site-specific integration. In some embodiments, polynucleotides are designed for gene editing using endonucleases such as CRISPR-cas systems, zinc finger nucleases, transcription activator-like effector nucleases (TALENs) and meganucleases.

[0534] Aspects of the present disclosure include vectors comprising polynucleotides. Any vector that can be used for gene delivery can be used. In some variants, viral vectors (such as AAV, adenovirus, lentivirus, retrovirus) are used. Non-limiting examples of vectors that can be used in the present disclosure include, but are not limited to, human immunodeficiency virus; HSV, herpes simplex virus; MMSV, Moloney murine sarcoma virus; MSCV, lentivirus, mouse stem cell virus; SFV, Semliki Forest virus; SIN, Sindbis virus; VEE, Venezuelan equine encephalitis virus; VSV, vesicular stomatitis virus; VV, and vaccinia virus.

[0535] In some embodiments, the vector is a lentiviral vector. Lentiviruses are a subclass of retroviruses. However, lentiviruses can integrate into the genome of non-dividing cells, while retroviruses can only infect dividing cells.

[0536] In some embodiments, the vector is a recombinant AAV vector. In some embodiments, the vector used in the methods of the present disclosure is encapsulated into a viral particle (e.g., an AAV viral particle, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16).

[0537] In some embodiments, the method comprises administering a vector comprising the polynucleotide or a pharmaceutical composition thereof.In some embodiments, the vector is used to deliver the polynucleotide to a target cell in vitro or in vivo.

[0538] In some embodiments, the method comprises administering a non-viral vector comprising a polynucleotide encoding a therapeutic agent, or a pharmaceutical composition thereof. In some embodiments, a non-viral vector or non-viral method is used to deliver a polynucleotide to a target cell in vitro or in vivo.

[0539] Non-limiting examples of non-viral delivery methods that can be used in the methods of the present invention to deliver polynucleotides encoding ABP include, but are not limited to: physical methods, needles, microprojectile gene transfer or gene guns, electroporation, sonoporation, photoporation, magnetofection, hydroporation, mechanical massage, chemical carriers, inorganic particles, calcium phosphate particles, magnetic particles, polymer carriers, or gene delivery agents such as silica, gold, cationic lipids, lipid nanoemulsions, solid lipid nanoparticles polyethyleneimine (PEI), chitosan, poly (DL-lactide) (PLA) and poly (DL-lactide-co-glycoside) (PLGA), dendrimers or polymethacrylates. Such methods can be found in Ramamoorth et al., (Ramamoorth et al., (2015) J. Clin. Diagnostic Res. 9(1): GE01-GE06 and Sung et al., (Sung et al., (2019) Biomaterials Research 23(8), pgs 1-87), which are incorporated herein by reference in their entirety.

[0540] In some embodiments, the method further comprises the step of detecting the presence / absence or level of a tumor-specific antigen in a biological sample of the subject before or after administration of the therapeutic agent, as described in Section 3.4. In some embodiments, the treatment methods provided herein are used to treat subjects diagnosed with MD or AL using the methods described in Section 3.4. In some embodiments, the diagnostic methods described in Section 3.4 are used to check the efficacy of the treatment methods provided herein. In some embodiments, the method comprises the step of determining the presence or absence of myeloid disorder (MD) and acute leukemia (AL) blasts in a sample obtained from a subject.

[0541] In some embodiments, the methods of the present disclosure include treating a subject with a myeloid disorder (MD) or acute leukemia (AL) comprising administering an effective amount of an ABP, an ABP-drug conjugate, a BiTE, a CAR, or an immune response cell comprising a CAR, or a pharmaceutical composition thereof.

[0542] In some embodiments, ABP, ABP-drug conjugates, BiTEs or immune response cells expressing CAR are administered in an amount sufficient to affect the survival of cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In certain embodiments, ABP, ABP-drug conjugates, BiTEs or immune response cells expressing CAR are administered in an amount sufficient to eliminate cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, ABP, ABP-drug conjugates, BiTEs or immune response cells expressing CAR are administered in an amount sufficient to reduce or kill cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, ABP, ABP-drug conjugates, BiTEs or immune response cells expressing CAR are administered in an amount sufficient to reduce or kill cancer cells expressing CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR-expressing immune response cell is administered in an amount sufficient to reduce or kill cancer cells in a subject.

[0543] In some embodiments, the effective amount of the immune response cells expressing CAR is 100,000 cells / kg to 15 million cells / kg. In some embodiments, the method includes administering immune response cells expressing CAR to a subject at a dosage of 100,000 cells / kg to 25 million cells / kg.

[0544] In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR comprises an antigen binding domain that binds to the target protein with a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D Binds to target protein.

[0545] In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR has a K of less than 500 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM as measured by biolayer interferometry. DBinds to human CD63, CD151, CD72, CD84, CD69 or CD109. In some embodiments, the ABP, ABP-drug conjugate, BiTE or CAR comprises an antigen binding domain that binds to human CD63, CD151, CD72, CD84, CD69 or CD109, with a K of D Less than 500 nM, 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM or 0.1 nM.

[0546] In some embodiments, ABP, ABP-drug conjugates, BiTE or CAR include commercially available antibodies obtained by affinity maturation or their modified antigen binding domains. In some embodiments, commercially available antibodies are antibodies for CD69 (e.g., Invitrogen 14-0699-82, ab201570, R&D MAB2359). In some embodiments, commercially available antibodies are antibodies for CD63 (e.g., ab59479, BD556019). In some embodiments, commercially available antibodies are antibodies for CD151 (e.g., Invitrogen MA5-16443, BD 556056). In some embodiments, commercially available antibodies are antibodies for CD84 (e.g., Biolegend 326002, NOVUS NBP2-44345). In some embodiments, commercially available antibodies are antibodies for CD109 (e.g., R&D MAB4385, BD56019). In some embodiments, the commercially available antibody is an antibody against CD72 (e.g., Biolegend 316202, BD555917, MAB 5405, Invitrogen PAS-97567).

[0547] In some embodiments, the ABP, ABP-drug conjugate, BiTE, or CAR comprises an antigen binding domain of A1, C1, F1, G1, C2, F2, H2, H3, F12, or B8, or a modification thereof.

[0548] 3.5.1 Other agents

[0549] In some embodiments, the treatment methods provided herein also include the combined administration of additional agents and ABP or its pharmaceutical composition, ABP-drug conjugate or its pharmaceutical composition, BiTE agent or its pharmaceutical composition, or immune response cells expressing CAR or its pharmaceutical composition. In some embodiments, additional agents and ABP or its pharmaceutical composition, ABP-drug conjugate or its pharmaceutical composition, BiTE or its pharmaceutical composition, or immune response cells expressing CAR or its pharmaceutical composition are administered separately, sequentially or together.

[0550] In some embodiments, ABP or its pharmaceutical composition is administered in combination with another agent. In some embodiments, the immune response cell expressing CAR or its pharmaceutical composition is administered in combination with another agent. The combined administration of ABP or its pharmaceutical composition combination with another agent may include administering another agent before, during, or after administering ABP or a pharmaceutical composition. In some embodiments, the combined administration of ABP or its pharmaceutical composition combination with another agent includes administering another agent before administering ABP or its pharmaceutical composition. In some embodiments, the combined administration of ABP or its pharmaceutical composition combination with another agent includes administering another agent after administering ABP or its pharmaceutical composition. In some embodiments, the combined administration of ABP or its pharmaceutical composition combination with another agent includes administering another agent simultaneously with administering ABP or its pharmaceutical composition.

[0551] In some embodiments, the additional agent is a chemotherapeutic agent or a biologic agent.

[0552] In certain embodiments, the additional agent is a chemotherapeutic agent. In specific embodiments, the chemotherapeutic agent is selected from cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacitidine, decitabine, and CPX-351 Composed of groups.

[0553] In specific embodiments, the chemotherapeutic agent is fludarabine.In other specific embodiments, the chemotherapeutic agent is cyclophosphamide.

[0554] In certain embodiments, the additional agent is a biologic agent. In some embodiments, the biologic agent is an antibody to a target protein other than CD63, CD151, CD72, CD84, CD69, or CD109.

[0555] In some embodiments, the additional agent is a Hedgehog signaling pathway inhibitor. In some embodiments, the Hedgehog signaling pathway inhibitor is a Sonic Hedgehog signaling pathway inhibitor. In some embodiments, the Sonic Hedgehog signaling pathway inhibitor is selected from Vismodegib, Sonidegib, and Arsenic Trioxide (ATO). In certain embodiments, the Hedgehog signaling pathway inhibitor is Daurismo TM ).

[0556] In some embodiments, the additional agent is a FMS-like tyrosine kinase 3 (FLT3) inhibitor. In certain embodiments, the FLT3 inhibitor is selected from midostaurin. Gilteritinib The group consisting of sorafenib, lestaurinib, quizartinib, and clelanib.

[0557] In some embodiments, the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor. In certain embodiments, the IDH1 or IDH2 inhibitor is ivosidenib Ensidipine

[0558] In some embodiments, the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor. In certain embodiments, the BCL2 inhibitor is venetoclax.

[0559] In some embodiments, the additional agent is a CD33 targeting agent. In certain embodiments, the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg TM ) or tariline-vadatuximab (SGN-CD33A).

[0560] In some embodiments, the additional agent is a cell cycle checkpoint inhibitor. In some embodiments, the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor.

[0561] In some embodiments, the additional agent is an immune checkpoint inhibitor. In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0562] 3.6 Pharmaceutical Compositions

[0563] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antigen binding protein (ABP) as described herein, an ABP-drug conjugate, a BiTE, or an immune response cell expressing a chimeric antigen receptor (CAR). Such compositions include an effective amount of an antigen binding protein (ABP), an ABP-drug conjugate, a BiTE, or an immune response cell expressing a chimeric antigen receptor (CAR) and a mixture of a pharmaceutically acceptable excipient.

[0564] In one aspect, the present disclosure includes a pharmaceutical composition comprising an ABP and a pharmaceutically acceptable excipient.

[0565] In another aspect, the disclosure includes a pharmaceutical composition comprising an ABP-drug conjugate and a pharmaceutically acceptable excipient.

[0566] In another aspect, the present disclosure includes a pharmaceutical composition comprising a BiTE agent and a pharmaceutically acceptable excipient.

[0567] In another aspect, the present disclosure includes a pharmaceutical composition comprising an immune response cell expressing a chimeric antigen receptor (CAR) and a pharmaceutically acceptable excipient.

[0568] Pharmaceutical compositions may contain formulation materials used to alter, maintain or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition.

[0569] Suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); pigments; flavorings and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone) ; low molecular weight polypeptide; salt-forming counterion (such as sodium); preservative (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvent (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohol (such as mannitol or sorbitol); suspending agent; surfactant or wetting agent (such as pluronics, PEG, sorbitol ester, polysorbate, such as polysorbate 20, polysorbate 80, triton, trimethylamine, lecithin, cholesterol, tyloxapal); stability enhancer (sucrose or sorbitol); extender (such as alkali metal halide (preferably sodium chloride or potassium chloride, mannitol sorbitol)); delivery vehicle; diluent; excipient and / or pharmaceutical adjuvant. Neutral buffered saline or saline mixed with the same serum albumin is a suitable diluent. Preservatives such as benzyl alcohol may also be added according to appropriate industry standards. The composition may be formulated as a lyophilized solution using an appropriate excipient solution (such as sucrose) as a diluent. Suitable ingredients are non-toxic to the recipient at the dosage and concentration used.

[0570] Optionally, the composition further comprises one or more additional agents, for example physiologically active agents, such as anti-angiogenic substances, chemotherapeutic substances (such as capecitabine, 5-fluorouracil or doxorubicin), analgesic substances, etc., non-exclusive examples of which are provided herein.

[0571] In another embodiment of the present disclosure, the compositions disclosed herein can be formulated into neutral or salt forms. Exemplary pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), and acid addition salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid), etc. The salts formed with the free carboxyl groups can also come from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount.

[0572] Carriers may also include any and all solvents, dispersion media, carriers, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and formulations for pharmaceutically active substances is well known. Supplementary active ingredients may also be added to the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0573] Those skilled in the art will determine the optimal pharmaceutical composition based on, for example, the intended route of administration, the delivery form, and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. These ingredients may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide. For example, a suitable composition may be water for injection, a physiological saline solution for parenteral administration.

[0574] In some embodiments, the active ingredient (e.g., ABP, ABP-drug conjugate) is present in the pharmaceutical composition at a concentration of at least 0.01 mg / ml, at least 0.1 mg / ml, at least 0.5 mg / ml, or at least 1 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, or 25 mg / ml. In certain embodiments, the active ingredient is present in the pharmaceutical composition at a concentration of at least 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, or 50 mg / ml.

[0575] In some embodiments, the pharmaceutical composition comprises one or more additional active ingredients in addition to a protein or polypeptide of the disclosure. The other active ingredients are one or more other active agents as described in 3.5.1.

[0576] The pharmaceutical composition can be formulated for administration by any route of administration suitable for human or veterinary medicine. In some embodiments, the pharmaceutical composition is suitable for injection. In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, intraperitoneal or subcutaneous administration. In some embodiments, the pharmaceutical composition is suitable for intravenous infusion. In some embodiments, the pharmaceutical composition is formulated for intrathecal or intracerebroventricular administration.

[0577] In some embodiments, the dosage range of the pharmaceutical composition is 100,000 cells / kg to 25 million cells / kg. In some embodiments, the dosage range of the pharmaceutical composition is 100,000 cells / kg to 15 million cells / kg. In various embodiments, the dosage range of the pharmaceutical composition is 100,000 cells / kg to 10 million cells / kg. In various embodiments, the dosage range of the pharmaceutical composition is 100,000 cells / kg to 25 million cells / kg. In some embodiments, the dosage range of the pharmaceutical composition is 300,000 cells / kg to 10 million cells / kg. In some embodiments, the dosage range of the pharmaceutical composition is 300,000 cells / kg to 25 million cells / kg.

[0578] In various embodiments, the unit dosage form is a vial, an ampoule, a bottle or a prefilled syringe. In various embodiments, the unit dose is administered in a drip bag. In some embodiments, the unit dosage form contains at least 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 25 mg, 50 mg, 75 mg or 100 mg of ABP or ABP-drug conjugate. In some embodiments, the unit dosage form contains at least 125 mg, 150 mg, 175 mg or 200 mg of ABP or ABP-drug conjugate. In some embodiments, the unit dosage form contains at least 250 mg of ABP or ABP-drug conjugate. In some embodiments, the unit dosage form contains at least 1×10 4 , 1×10 5 , 1×10 6 , 1.5×10 6 , 1×10 7 , 2×10 6 , 2.5×10 6 , 2.5×10 7 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5×10 7 , 1×10 8 , 2.5×108 , 5×10 8 , 7.5×10 8 , 1×10 9 , 2.5×10 9 , 5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , or 1×10 9 In some embodiments, the unit dosage form contains at least 1.5×10 4 , 1.5×10 5 , 1.5×10 6 , 1.5×10 7 , 2.5×10 7 , 5×10 7 , 1×10 8 , 2.5×10 8 , 5×10 8 , 7.5×10 8 , 1×10 9 , 2.5×10 9 , 5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , or 1×10 9 CAR-expressing immune response cells.

[0579] In typical embodiments, the pharmaceutical composition in the unit dosage form is in liquid form. In various embodiments, the unit dosage form contains 0.1 mL to 50 mL of the pharmaceutical composition. In some embodiments, the unit dosage form contains 1 mL, 2.5 mL, 5 mL, 7.5 mL, 10 mL, 25 mL, or 50 mL of the pharmaceutical composition.

[0580] In certain embodiments, the unit dosage form is a vial containing 1 ml of a pharmaceutical composition having a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml. In some embodiments, the unit dosage form is a vial containing 2 ml of a pharmaceutical composition having a concentration of 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, or 1 mg / ml.

[0581] In some embodiments, the pharmaceutical composition in unit dosage form is in a solid form, such as a lyophilisate suitable for dissolution.

[0582] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect.

[0583] 4. Examples

[0584] 4.1 Example 1: Identification of tumor-specific antigens

[0585] Identification of tumor-specific antigens (TSAs) from AML patient samples, such as Figure 1 as outlined.

[0586] In brief, candidate genes for tumor-specific antigens are obtained from computer simulation target antigen discovery analysis by applying precise selection methods. According to the gene expression profile for risk stratification previously generated from 85 children's AML samples (GSE75461) by HTA 2.0 (Affymetrix) platform, genes overexpressed in AML primitive cells are selected. HTA2.0 (Affymetrix) platform maximizes the collection of valuable information by minimizing the conserved sequences synthesized on the array. This high-resolution array design contains unprecedented> 6 million probes covering coding transcripts and non-coding transcripts. 70% of the probes on the array cover the exons of coding transcripts, and the remaining 30% of the probes on the array cover exon-exon splicing junctions and non-coding transcripts. The unprecedented coverage of the array provides insights into all available coding and non-coding transcripts.

[0587] The data were used to identify highly expressed genes, i.e. genes whose expression was above the upper inflection point of the distribution (>3.4) in all 85 AML samples but not in bone marrow healthy control samples (overexpressed genes in the AML patient cohort and limma controls of 3 HTA-arrays were obtained from three bone marrow samples from pediatric healthy donors after Benjamini-Hochberg multiplicity correction).

[0588] Of these genes, a subset was selected based on the validated surface omics protein dataset http: / / wlab.ethz.ch / cspa / when they were cell surface protein antigens (CSPA). Candidates from this process were then validated using the public dataset TARGET database, from which the gene expression matrix of 262 AML pediatric samples was downloaded. TARGET data were RMA normalized; 85 Italian AML patient cohorts and 262 TAGET AML samples were z-score normalized so that the relative expression of the selected genes could be compared by box plots between the two cohorts. This allowed the identification of 44 genes (first list - FL) as the best representative surface antigens of childhood AML at disease onset, which are not expressed in healthy donor bone marrow.

[0589] This first list - FL's 44 candidate genes were then analyzed for broad coverage and specificity by querying public databases, with the goal of selecting the most promising targets for in vitro testing of the following protein expression. GeneCards software was used to view TSA functional descriptions, gene chromosomal localization, associations with known pathologies / cancers, mRNA expression in normal human tissues, estimated protein expression, subcellular locations, and pathways involved; Pubmed was used to query TSAs for previous involvement in cancers including AML and to exclude their involvement in previous CAR-T development projects, and XenaBrowser was used to predict the expression selectivity of candidate TSAs in AML. This is an online platform for exploring 1500 cancers including + Datasets and 50 + Public and private multigenes and clinical / phenotypes for 100 cancer types. The tool provides interactive online visualization of 100 cancer genomics datasets, including data from The Cancer Genome Atlas (TCGA), the International Cancer Genome Consortium (ICGC), the Genomic Data Community (GDC), and the UCSC RNA-seq Compendium. Xena supports nearly all functional genomic data, including SNVs, INDELs, large structural variants, copy number variants, genes, transcripts, exons, miRNAs, LncRNAs, protein expression, DNA methylation, ATAC-seq signals, phenotypic annotations, and higher-level derived genomic parameters. The refinement process applied as inclusion criteria gene function (cancer-related) and localization on the cell surface, and as exclusion criteria i) considered functions of enzymes and ribosomal components, ii) localization other than the cell surface, including nuclear membrane; iii) previous participation in AML diagnostic panels or CAR-T development, iv) previously part of patents viewed Espacenet, trademark information, patent scope, and v) high documented expression in multiple human tissues. At the end of this refinement process, 26 of the 44 genes in FL were discarded, providing 18 candidate TSAs.

[0590] Applicants then generated a second list - SL - by querying the same computer simulation data set and selecting those genes whose expression was below the inflection point of the distribution (>2.4 and <3.4). This new list included an additional 32 candidate TSAs that underwent the same analytical process as the above TSA refinement. Due to exclusion criterion (iv), 24 of the 32 TSAs identified in SL were discarded, providing an additional 8 candidate TSAs.

[0591] These 26 candidate TSAs (18 candidate TSAs from FL and 8 additional candidate TSAs from SL) were then characterized for protein expression and localization by flow cytometry (FC). Primary commercial antibodies that recognized the candidate TSAs were first tested with positive control cells. Once the antibodies were demonstrated to provide staining on the control, cell surface staining of the TSAs was performed with a minimum of 2 AML cell lines (SHI-1 and HL-60). 11 of the 26 candidate TSAs were confirmed to be expressed on the surface of AML cells (e.g., 10 cells / ml) by flow cytometry. Figure 1 strong or weak expression as indicated).

[0592] Six AML-TSAs (CD63, CD151, CD72, CD84, CD69, and CD109) were finally selected based on the strong positive signals and specificity detected by flow cytometric analysis with AML cell lines SHI-1 and HL-60 ( Figure 1 For further validation, their expression on additional AML cell lines including KASUMI-1, MOLM-13 and MV4-11 was detected by immunofluorescence using the same antibodies used for flow cytometry analysis.

[0593] Flow cytometric analysis and fluorescent immunohistochemistry data are provided in Figure 2A and 2B (CD69), Figure 3A and 3B (CD63), Figure 4A and 4B (CD151), Figure 5A and 5B (CD84), Fig. 6A and 6B (CD109) and Fig. 7A and 7B (CD72). Figure 2C (CD69), Figure 3C (CD63), Figure 4C (CD151), Figure 5C (CD84), Figure 6C (CD109) and Figure 7C (CD72), the expression of the antigen was specific for AML cells - much lower or no expression was detected by flow cytometric analysis of healthy hematopoietic cells - fractionated subsets obtained from healthy donor peripheral blood mononuclear cells (PBMCs) included CD19 + B lymphocytes, CD3 + T lymphocytes, CD33 + Bone marrow progenitor cells, and CD34 derived from umbilical cord blood samples + Cells. In healthy BM subsets, in normal regenerating bone marrow, in normal regenerating bone marrow, CD34+ and CD34 + TSA expression in the CD38- subset, in gated blasts of acute lymphoblastic leukemia patients, in gated blasts of AML patients at diagnosis, in gated blasts of AML patients at relapse, in gated blasts of AML patients with residual blasts, and in gated blasts of patients with other malignancies, such as patients with myeloid tumors, is also summarized in Table 18 below, respectively.

[0594] Table 1: Expression of selected TSAs in healthy blood monocyte subsets

[0595] Table 1 provides the expression of TSA in healthy blood samples and shows the heterogeneous dim expression of TSA in peripheral blood monocyte subsets.

[0596]

[0597] Strong positive (WHO classification) Weak positive (WHO classification)

[0598] POS B: positive bright POS D: positive dim

[0599] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0600] POS H: Positive heterogeneity

[0601] PP2: Partially positive 2 (AIEOP-BFM classification)

[0602] Table 2: Expression of selected TSA subsets in normal regenerating bone marrow

[0603]

[0604] Strong positive (WHO classification) Weak positive (WHO classification)

[0605] POS B: positive bright POS D: positive dim

[0606] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0607] POS H: Positive heterogeneity

[0608] PP2: Partially positive 2 (AIEOP-BFM classification)

[0609] Table 2 provides TSA expression in healthy subpopulations from regenerating bone marrow collected at the end of treatment, and showed low / dim expression in subpopulations of the myeloid lineage in 3 / 6 TSAs.

[0610] Table 3: CD34 in normal regenerating bone marrow + and CD34 + CD38 - Expression of selected TSAs in subpopulations

[0611]

[0612] Strong positive (WHO classification) Weak positive (WHO classification)

[0613] POS B: positive bright POS D: positive dim

[0614] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0615] POS H: Positive heterogeneity

[0616] PP2: Partially positive 2 (AIEOP-BFM classification)

[0617] Table 3 provides the CD34 stem cell precursors from the regenerating bone marrow collected at the end of treatment. + CD38 - TSA expression in subpopulations and showed weak expression of some TSAs.

[0618] Table 4: TSA expression in B or T acute lymphoblastic leukemia at diagnosis

[0619]

[0620] Strong positive (WHO classification) Weak positive (WHO classification)

[0621] POS B: positive bright POS D: positive dim

[0622] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0623] POS H: Positive heterogeneity

[0624] PP2: Partially positive 2 (AIEOP-BFM classification)

[0625] Table 4 provides TSA expression in acute lymphoblastic leukemia samples at diagnosis and shows that all TSAs are suitable for detecting B lymphoblasts, while T-lymphoblasts only express CD69 and CD84.

[0626] Table 5: TSA expression in AML at diagnosis

[0627]

[0628] Strong positive (WHO classification) Weak positive (WHO classification)

[0629] POS B: positive bright POS D: positive dim

[0630] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0631] POS H: Positive heterogeneity

[0632] PP2: Partially positive 2 (AIEOP-BFM classification)

[0633] Table 5 provides TSA expression in acute myeloid leukemia samples at diagnosis and shows that all TSAs are suitable for detecting bone marrow blasts, with CD69 and CD84 being the most specific.

[0634] Table 6: TSA expression in AML at relapse

[0635]

[0636] Strong positive (WHO classification) Weak positive (WHO classification)

[0637] POS B: positive bright POS D: positive dim

[0638] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0639] POS H: Positive heterogeneity

[0640] PP2: Partially positive 2 (AIEOP-BFM classification)

[0641] Table 6 provides TSA expression in acute myeloid leukemia samples at diagnosis, showing that all TSAs are suitable for detecting blasts at disease relapse, with CD69 and CD84 being more specific.

[0642] Table 7: TSA expression in AML treated with residual blasts (<5%)

[0643]

[0644] Strong positive (WHO classification) Weak positive (WHO classification)

[0645] POS B: positive bright POS D: positive dim

[0646] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0647] POS H: Positive heterogeneity

[0648] PP2: Partially positive 2 (AIEOP-BFM classification)

[0649] Table 7 provides TSA expression in bone marrow samples collected during treatment with residual disease in the bone marrow of 0-5% blasts, with CD69 and CD84 being the most suitable for detecting low disease levels.

[0650] Table 8: TSA expression in myeloid neoplasms

[0651]

[0652] Strong positive (WHO classification) Weak positive (WHO classification)

[0653] POS B: positive bright POS D: positive dim

[0654] POS M: Positive Moderate PP1: Partially Positive 1 (AIEOP-BFM classification)

[0655] POS H: Positive heterogeneity

[0656] PP2: Partially positive 2 (AIEOP-BFM classification)

[0657] Table 8 provides TSA expression in bone marrow samples of myeloid neoplasms at diagnosis and shows the ability of CD69, CD84, and CD72 to reveal dysplastic cells.

[0658] Specific expression was further confirmed by observing no or low TSA expression in primary fibroblasts from healthy donors ( Figure 8 ).

[0659] The expression of TSA was also tested in other cancer cell lines representing solid and liquid tumors other than AML ( Fig.10 ). Six TSAs were expressed at different levels, but the overall expression level was low in these other cancer cells.

[0660] These studies demonstrated that the six selected TSAs were strongly and selectively expressed in most, if not all, AML cell lines tested. Overexpression of the six TSAs was also assessed and confirmed using two AML patient-derived explants (PDXs) available in the laboratory, generated from primary pediatric AML samples, by interrogating their gene expression profiles.

[0661] The stability and predictive value of TSA were studied by evaluating the mRNA expression of TSA in childhood AML samples obtained for diagnosis or for testing remission after treatment, according to the genetic risk stratification. TSA overexpression was confirmed in the active phase of the disease (at diagnosis), and expression was reduced in samples collected at the end of treatment.

[0662] method

[0663] Patient samples and cell lines. Bone marrow (BM) samples were collected, processed, and analyzed according to standard operating procedures. 42 samples from de novo AML patients—30 at diagnosis, and 12 at AML relapse. Four samples from bone marrow tumors, 7 AML samples collected during treatment with residual blasts (<5%), 6 B-ALL samples and 1 T-ALL sample at diagnosis, and 13 BM samples collected during follow-up when the disease was in remission and had regenerative features as previously defined (Buldini B. et al., BJH 2018).

[0664] All AML cell lines were purchased from DSMZ. HL-60, Kasumi-1 and MOLM-13 were cultured in RPMI (GIBCO, Life Technologies, Paisley, UK) supplemented with 1% penicillin-streptomycin (GIBCO, Life Technologies, Grand Island, NY, USA), 1% L-glutamine (GIBCO) and 10% fetal bovine serum (GIBCO). MV4-11 and SHI-1 were cultured in Dulbecco's modified medium (GIBCO) supplemented with 1% penicillin-streptomycin, 1% L-glutamine and 10% fetal bovine serum.

[0665] Peripheral blood mononuclear cells (PBMC) were obtained from the buffy coat of informed consent healthy donors by Ficoll density separation. According to the protocol, CD34 positive cells were obtained from the cord blood of informed consent healthy donors by Ficoll density separation and then magnetic separation in the column of CD34 MicroBead Kit (MiltenyiBiotech GmbH, Bergish Gladbach Germany). Primary cells from whole blood (maximum 100 μL per tube) were stained with conjugated antibodies.

[0666] Flow cytometric analysis

[0667] BM samples were stained with primary conjugated antibodies for 15 minutes in the dark, then hemolyzed and centrifuged. Cells were resuspended in PBS and analyzed by BD FACS CANTO II, acquiring a minimum of 30,000 events.

[0668] Primary antibodies used for clinical multiparameter analysis (Table 4) were CD69 A488 (R&D Systems, catalog no. FAB23591G), CD63 PE (BD Pharmingen, catalog no. 556020), CD 151 PE (R&D Systems, catalog no. FAB1884P), CD84 APC (Biolegend, catalog no. 326010), CD109 A488 (R&D Systems, catalog no. FAB4385G), CD72 BV421 (BD Pharmingen, catalog no. 743794), together with the following anti-human antibodies: CD45 V500 (BD Pharmingen, catalog no. 560777), CD45 APC-Cy7 (BD Pharmingen, catalog no. 348815), CD34 PC5 (Beckman Coulter, catalog no. A07777), CD38 PE-Cy7 (BD Pharmingen, catalog number 335825), CD33 APC (BD Pharmingen, catalog number 551378), CD7 V450 (BD Pharmingen, catalog number 642916), HLA-DRV500 (BD Horizon, catalog number 561224), CD33 PC5 (Beckman Coulter, catalog number I112647), CD117 PC7 (Beckman Coulter, catalog number IM3698), CD34 A750 (Beckman Coulter, catalog number B92463), CD38 V450 (BD Pharmingen, catalog number 561378) CD19 PE (exbio, catalog number 1P-305-T100), CD7 PC5 (Beckman Coulter, catalog number I113613), CD34 APC (BD Pharmingen, catalog number 345804), CD3 APC-Cy7 (BD Pharmingen, catalog number 341110).

[0669] Table 9: Groups detected for TSA expression by multiparameter flow cytometry analysis

[0670] FITC PE PC5 PC7 APC APCCY7 V450 V500 1 CD69 CD63 CD34 CD38 CD33 CD45 CD7 HLA-DR 2 CD109 CD151 CD33 CD117 CD84 CD34 CD38 CD45 3 CD69 CD19 CD7 CD38 CD34 CD3 CD72 CD45 4 CD69 (CD4) CD7 CD34 CD84 CD45 CD38 CD8

[0671] Table 9 shows a diagnostic panel of antibodies used to determine the immunophenotype when diagnosing acute leukemia or myeloid neoplasms by flow cytometry.

[0672] Intracellular staining for antibody validation

[0673] All primary antibodies listed above were first validated by performing intracellular staining of PBMCs as recommended by the guidelines.

[0674] Briefly, cells (6×10 5 ) were fixed with 4% paraformaldehyde in phosphatase buffered saline (PBS) at room temperature for 15 minutes, and then rinsed three times with PBS. Next, they were permeabilized with 0.1% TWEEN20 in PBS for 20 minutes and blocked with 3% bovine serum albumin (BSA) in PBS for 30 minutes. The permeabilized cells were stained with primary antibodies and Fc receptor blocking (Miltenyi) for 30 minutes, and then incubated with secondary antibodies for 20 minutes.

[0675] Cell surface staining

[0676] The primary antibodies were tested for binding on the surface of AML and other cancer cell lines, PBMCs and CD34 positive cells from umbilical cord blood. Cells (3×10 5 ) and stained with primary antibody and Fc receptor blocking for 20 minutes. After washing with PBS, cells were stained with secondary antibody for 20 minutes.

[0677] For PBMC subsets, antibodies against TSA were used with the following anti-human antibodies for 20 minutes: CD3-APC (Beckam Coulter, Marseille, France), CD19-PE (Beckam Coulter), CD33-PC5 (Beckam Coulter). Next, cells were stained with secondary antibodies for 20 minutes. Cells were analyzed using CytoFLEX (Beckam Coulter) and Flow Jo software (version 9.7, TreeStar Inc.).

[0678] Immunofluorescence

[0679] The primary antibodies used for these analyses were from eBioscience, Abcam, and BD; the secondary antibody was goat anti-mouse IgG Alexa488 antibody (Life Technologies). 5) were seeded onto the bottom of chamber slides (FALCON, BigFlats, NY, USA) pre-coated with fibronectin (40ug / ml) (Corning) for 2 hours at 37°C. After this incubation time, the cells were stained with MemBrite Fix dye (Biotium), a special membrane dye, for 5 minutes at 37°C according to the instructions. The cells were fixed with 4% formaldehyde in PBS for 15 minutes, washed, and blocked with 3% BSA in PBS for 30 minutes. After saturation, the cells were stained with primary antibodies and Fc receptor blocking at 4°C overnight, and then stained with secondary antibodies for 1 hour at room temperature. The cells were imaged using a Zeiss2.6 laser scanning microscope. Images were analyzed using Image J win 32 software.

[0680] 4.2 Example 2: Treatment Methods Targeting AML-TSA

[0681] Six AML-TSAs (i.e., CD63, CD151, CD72, CD84, CD69, and CD109) were tested as described in Example 1 and selected based on i) strong and widespread expression in primary myeloid tumors, acute leukemias, and acute myeloid leukemias, ii) expression in CD34 + CD38 + The proteins were selected based on their absence / low expression in HSPCs, iii) association with tumorigenesis, and iv) expression in AML cell lines.

[0682] To develop a therapy targeting one of the TSAs, 2 to 4 different commercial antibodies against each target were tested to design the best single-chain variable fragment (ScFv) against each TSA. For each antibody, the results of flow cytometric analysis on AML cell lines are provided in Figure 8 middle.

[0683] In addition, new antibodies against TSA are generated using methods known in the art, such as using steps involving immunogen preparation, immunization, hybridoma production, screening and purification, or natural or immune antibody libraries for panning phage display. The new antibodies are also tested for binding affinity and specificity to the TSA target.

[0684] The antibodies selected according to the binding affinity and specificity of the antibodies are used to develop ABP-drug conjugates and CAR-T cells. ABP-drug conjugates are produced by conjugating the selected antibodies to cytotoxic agents. CAR is produced by using the antigen binding domain of the selected antibody as the extracellular domain. A polynucleotide encoding CAR is produced, the CAR comprising an antigen binding domain, a transmembrane domain, a signaling domain and optionally at least one co-stimulatory domain. The polynucleotides are transfected into T cells or NK cells to produce CAR-T cells or CAR-NK cells.

[0685] Each of the ABPs, ABP-drug conjugates, and CAR-T cells were tested in vitro using AML cell lines as well as primary pediatric AML samples. When the ABPs, ABP-drug conjugates, or CAR-T cells were applied to cell cultures of AML cell lines or primary AML cells, they induced cytotoxic effects specifically against cancer cells.

[0686] ABP-drug conjugates and CAR-T cells were also tested in vivo using NSG (NOD / scid IL-2Rgnull) mice (a syngeneic human disease model) injected intravenously with AML cell lines and by using patient-derived xenograft (PDX) animal models. Cancer cells in the animal models were reduced after administration of ABP, ABP-drug conjugates, or CAR-T cells in the peripheral blood, bone marrow, or spleen.

[0687] 4.3 Example 3: Treatment Methods Targeting AML-TSA Using CAR-T Cells

[0688] Based on the use of the generated CAR constructs, the use of selected TSAs (CD63, CD151, CD72, CD84, CD69 and CD109) as targets for T cell immunotherapy approaches was validated.

[0689] Six tumor-associated antigens, namely CD69, CD63, CD151, CD84, CD109 and CD72, were identified as being uniquely associated with childhood acute leukemia and bone marrow diseases. The CD69 and CD84 antigens were characterized in vitro and in vivo, and were confirmed to be highly specific for acute myeloid leukemia (AML) primary samples collected at diagnosis and relapse. It was found that the CD69 and CD84 antigens were highly expressed in bone marrow disorders, including acute myeloid leukemia (AML) bone marrow samples collected at diagnosis (in 75% and 96% of cases, respectively) or relapse, and samples collected after treatment with residual disease defined as primitive cells>1% and ≤10%. Interestingly, CD72 is expressed in the majority of B cell precursor acute lymphoblastic leukemia samples examined at diagnosis (98% of BCP-ALL) and in most AML cases (66%) at diagnosis.

[0690] Based on this data, 11 new single-chain variable fragment (ScFv) sequences (A1, C1, F1, G1, C2, F2, H2, H3, F12, and B8) were developed by phage display for the generation of chimeric antigen receptors (CARs) that recognize CD69 or CD84, or a combination of CD69 and CD84, on AML blasts. The CD72 antigen was shown to be highly specific for acute lymphoblastic leukemia (ALL) and has great potential for the diagnosis of ALL patients treated with CD19-directed immunotherapy, who often lose the CD19 marker. Design and preparation of novel CD84 and CD69 CAR-T cells

[0691] Two new antigens, CD84 and CD69, were prioritized as potential targets for AML immunotherapy. Phage display was used to discover high-affinity antigen binders to the two antigens from a large combinatorial library containing up to billions of antibody targets. The six-round panning method was considered an iterative process of enriching phages within a phage population that had high binding affinity to our target compared to other targets by exposing the library to our antigens after enriching the phage population with high affinity, and then eluting and amplifying only those phages with the highest binding affinity. This qualitative selection method identified 87 CD69 clones and 6 CD84 clones. Validation using ELISA technology, which allows for more precise assessment of antibody-antigen interactions by quantitative immunoassays, confirmed that 8 ScFv sequences and 2 ScFv sequences were good candidates for CD69 and CD84, respectively. Ten new ScFv genes were generated by gene synthesis and then cloned into a third-generation chimeric antigen construct (CAR) containing the CD28 H / TM and 4-1BB co-stimulatory domains and the ζ (CD3ζ) signaling domain into the pUC57 plasmid. The CAR was cut and pasted into a third-generation lentiviral transfer plasmid by restriction endonuclease technology for the production of high-titer lentiviral vectors (LV) ( Fig.13 ). The workflow then includes isolation of healthy donor T cells, followed by efficient activation, gene transfer of the CAR construct into activated T cells, CAR-T cell expansion, and phenotypic analysis of the final CAR-T cell product alone (---) or after co-culture with the target AML cell line.

[0692] CAR-T cells were prepared using four different specific ScFvs identified in a human natural antigen-binding fragment (Fab) phage library, i.e., 14722-P1-F12 and 14722-P1-B8 that recognize CD84, and 14721-P1-H3 and 14721-P1-G1 that recognize CD69. Figure 27 provides the nucleotide (SEQ ID NO: 24-34) and amino acid sequences (SEQ ID NO: 35-44) of the four CAR constructs. Prior to viral transduction, peripheral blood mononuclear cells (PBMCs) and T cells collected from healthy donors were separated by magnetic enrichment using CD4 and CD8 microbeads, and then transfected with T Cell TransAct TM The culture medium was activated. Then, in TexMACS TM After 48 h of expansion in medium supplemented with IL-7 and IL-15, T cells were transduced with CAR encoding LV at a multiplicity of infection (MOI) of 10. 24 h after transduction, the vector was washed away, and the CAR-T cells were expanded for 14 days ( Fig.14 ). Transduction efficiency was measured by digital droplet polymerase chain reaction (ddPCR) and expressed as vector copy number (VCN) / cell; the average VCN of all prepared CAR-T cells was between 2.5 and 33.7, and there was no significant difference in VCN between different CAR-T products ( Fig.15 ). Mock-transduced T cells (empty CAR) were used as control.

[0693] To appropriately test the potency and specificity of the newly prepared CD84 and CD69 CAR-T cells in vitro and in vivo, AML cell lines were first screened for CD84 and CD69 cell membrane expression by flow cytometry. HL-60 and SHI-1 were used as target cells, both of which expressed antigens at high levels ( Fig.16 ); MV4-11 and MOLM-13 cells only expressed CD84, while Kasumi-1, U937, and K562 cells only expressed CD69 ( Fig.16 ), as appropriate control cell lines for CD69 or CD84, respectively.

[0694] CAR design does not affect the expansion, viability, and phenotype of CD84 and CD69 CAR-T cells

[0695] At the end of the 14-day expansion (corresponding to day 17 of the CAR-T cell preparation process), the prepared CAR-T cells were counted and showed a 2-20-fold expansion rate ( Fig.17 ), which is highly variable mainly due to the starting donor PBMCs ( Fig.18A ). T cells are alive, in CD4 + Enrichment of more than CD8 + cell( Fig.18B), and most of them contain natural T cells and stem cell memory T cells (T n + T scm , CD197[CCR7] + CD45R0 - ) and central memory T cells (T cm , CD197(CCR7) + CD45R0 + ), as expected ( Fig.19A and 19B ). After expansion, CD84 and CD69 CAR-T cells exhibited the same degree of activation and exhaustion as empty CAR-T cells, as shown by upregulation of CD25, CD154, CD107a, and CD137, as well as CD366, CD223, and CD279 in response to culture conditions. Notably, identical results were observed on control T cells (empty CAR), confirming that the CAR expression cassette does not alter T cell function ( Fig. 20A and 20B ). CD84 and CD69 CAR-T cells have potent anti-tumor lytic activity in vitro

[0696] To test the activity of newly generated CD84 and CD69 CAR-T cells, CAR-T cells were maintained in culture medium alone (---) or co-cultured with target AML cell lines expressing the antigen. Significant in vitro antitumor activity against target cell lines was measured by the four ScFv sequences tested, ranging from 10-80% on HL-60 and 50-90% on SHI-2 cells. + The frequency of dead cells (Annexin V + and / or 7AAD + , Fig.21 ), 2) lysis efficiency ( Fig. 22 ) and 3) bioluminescent signals in luciferase-transduced AML cells ( Fig.23 ) further confirmed these results. The persistence of activated CAR-T cells was also monitored by cell counting for up to 48 hours in vitro. The activity of CAR-T cells was characterized by increased proliferation of T lymphocytes (black squares) and a decrease in the number of target AML cells (white squares, Fig.24 ).

[0697] Specific lytic activity was shown in vitro against AML target cell lines with A1, F1, C2, and H2 ScFvs targeting target cell lines expressing CD69. Specific lytic activity was shown in vitro against primary AML cells derived from childhood AML xenografts (PDXs) by CAR-T and anti-CD-84B8 and F12 ScFvs.

[0698] CD84 and CD69 CAR-T cells vs CD34 + Hematopoietic stem and progenitor cells (HSPC) show no toxicity

[0699] CD84 and CD69 CAR-T cells targeting CD34 + Potential off-target activity of hematopoietic stem and progenitor cells (HSPCs) was investigated in a standard colony forming unit (CFU) assay at a 1:1 effector:target ratio (E:T). Neither CD84 nor CD69 CAR-T constructs were active against CD34 + cells, as they neither induce CD34 + The number of CFU formed by HSPCs was significantly reduced and CD34 + Decreased viability ( Fig.25A and Fig.25B ).

[0700] T cells expressing CD84 and CD69 CARs recognize and kill AML cell targets in vivo

[0701] To test the in vivo activity of CAR-T cells, CAR-T cells targeting CD84 and CD69 were generated and infused into NOD / SCIDγ (NSG) mice transplanted with target AML cell lines. The AML cell lines were engineered to express luciferase for non-invasive bioluminescence imaging (BLI). Briefly, mice were injected with 0.5 × 10 6 AML cells / mouse (day 1), and then (day 3) injected with 1.5×10 6 CAR-T cells or mock-transduced T cells (MOI 10 empty CAR / mouse; n=6-8 for each tested condition). Mice treated with B8 and F12 (CD84) and G1 and H3 (CD69) CAR-T cells experienced a statistically significant reduction in leukemic burden assessed by BLI already at day 14 after AML challenge, which became even more pronounced over time (at days 23 and 35) ( Fig.26A , 26B, 26C).

[0702] Materials and methods

[0703] Human cell lines

[0704] HL-60 and SHI-1 AML cell lines were used as target cells for TSA screening. These cell lines were grown in RPMI (Life Technologies; 11875093) supplemented with 10% fetal bovine serum (FBS; Life Technologies; 10270106), 1% penicillin-streptomycin (P / S, 10000U / mL, Life Technologies; 15140148) and 1% L-glutamine (200mM, Thermo Fisher; 25030024), except for the SHI-1 cell line, which was cultured in DMEM (Life Technologies; 41965039) supplemented with 10% FBS and 1% P / S, and HEK293T was grown in IMDM (Euroclone; ECB2072L) supplemented with 10% FBS, 1% P / S and 1% L-glutamine. All cells were cultured at 37°C in a humidified incubator with 5% CO2.

[0705] clone

[0706] The CAR expression cassette (1506 bp) was cloned from a pUC57 vector (synthesized by ProteoGenix SA) into a third-generation LV transfer plasmid under the control of the human phosphoglycerate kinase promoter (hPGK). One Shot TOP10 chemically competent E. coli (ThermoFisher, Walthan, MA, USA) was transformed and DNA was extracted with a DNA Maxiprep Kit (Termo Fisher Scientific; K210017). The process was controlled by digestion with BamHI and SalI enzymes and agarose gel electrophoresis. Sanger sequencing of the plasmid was performed to verify the correct insertion of the expression cassette in the backbone.

[0707] CD4 from PBMC + and CD8 + Isolation and expansion of T cells

[0708] PBMCs were isolated from healthy donors by density gradient centrifugation using Lymphoprep (StemCell technologies; 07861); PBMCs, CD4+, and CD8+ cells were magnetically separated using an autoMACS instrument (Miltenyi Biotec; Bergisch Gladsbach, Germany) with CD4 and CD8 microbeads (Miltenyi Biotec; 130-045-101 and 130-045-201, respectively) according to the manufacturer's instructions. Isolated T cells were then activated on day 1 for expansion using TransAct (Miltenyi Biotec; 130-111-160) in TexMACS medium (Miltenyi Biotec; 130-097-196) containing 1% P / S and supplemented with recombinant human IL-7 (500 IU / ml) and IL-15 (84 IU / ml) (Miltenyi Biotec; 130-095-362 and 130-095-764, respectively). On day 4, cells were washed from TransAct and lentivirus and then incubated in TexMACS medium containing IL-7 and IL-15 at 1-2 × 10 6 cells / mL were maintained.

[0709] Lentiviral vector production and transduction of human T cells

[0710] LV was produced by transient transfection of HEK293T packaging cell line as described previously (Langford-Smith et al., 2012). Briefly, 70% confluent cells were co-transfected with Gag / PoL (III gen), Env and Rev packaging plasmids, pAdvantage plasmid (pADV) and transfer vector plasmid with expression cassette. After 48 hours from transfection, lentiviral supernatant was collected, ultracentrifuged, aliquoted and stored at -≤65°C.

[0711] On day 3, T cells were transduced with LV at an MOI of 10 using 0.01 mg / ml of Vectofusin-1 (Miltenyi Biotec; 130-111-163). After 16 hours of transduction, cells were washed from LV and TransACT. To evaluate transduction efficiency, the VCN of each cell was measured. On day 14 after transduction, total genomic DNA of transduced T cells was extracted using the Dneasy Blood & Tissue Kit (Qiagen; 69504). To determine the VCN of each cell, ddPCR was used with a reaction mixture containing a dUTP-free ddPCR Supermix probe (Bio-Rad; 1863024) and a primer-probe set for the target and reference genome (ddPCRTM CNV Assay [FAM] 10031277; ddPCRTM CNV Assay [HEX] 10031244). Droplets were prepared by Automated Droplet Generator (Bio-Rad) and read with QX200 droplet reader (Bio-Rad). VCN was analyzed with QuantaSoft droplet reader software and determined by the ratio of target gene concentration to reference gene concentration multiplied by the copy number of the reference gene in the reference genome. Activation, depletion, and differentiation markers analyzed by flow cytometry

[0712] Cells were stained with fluorescent dye-conjugated primary antibodies and subtype controls for 15 minutes at room temperature. The stained cells were washed and immediately analyzed using a FACSCelesta™ Cell Analyzer (BD Biosciences). The following antibodies were used:

[0713] CD3 (PE-Vio615, Miltenyi Biotec; 130-114-520), CD4 (VioGreen, Miltenyi Biotec; 130-113-230), CD8 (APC, Miltenyi Biotec; 130-110-681), CD45 (VioBlue, Miltenyi Biotec; 130-110-637), CD34 (PE, Miltenyi Biotec; 130-124-456), CD38 (APC, Miltenyi Biotec; 130-123-852), CD197 (CCR7), (VioBlue, Miltenyi Biotec; 130-117-353), CD45RO (APC, Miltenyi Biotec; 130-117-353) Biotec; 130-113-556), CD223 (VioBlue, Miltenyi Biotec; 130-118-549), CD279 (PE, Miltenyi Biotec; 130-120-385), CD366 (APC, Miltenyi Biotec; 130-119-781), CD154 (VioBlue, Miltenyi Biotec; 130-116-615), CD25 (PE, MiltenyiBiotec; 130-114-541), CD137 (APC, Miltenyi Biotec; 130-110-764), CD69 (PE, MiltenyiBiotec; 130-112-613), CD84 (APC, Biolegend; 326010) CD107a (PE, Miltenyi Biotec; 130-111-621) and CD33 (FITC, Miltenyi The data were analyzed using FlowJo software (BD Biosciences, v10).

[0714] In vitro cytotoxicity assay

[0715] At the end of T cell expansion (day 17), cytotoxicity was determined by co-culturing AML target cell lines (antigen positive) with CAR-T cells or untransduced T cells at an E:T ratio of 1:1 for 48 hours. The co-cultured cells were stained with Annexin V-PE (Miltenyi Biotec; 130-118-363) and 7-AAD Staining Solution (Miltenyi Biotec; 130-11-568) and analyzed by flow cytometry using FACSCelestaTM Cell Analyzer and FlowJo software. The percentage of cell lysis was calculated using the following formula:

[0716]

[0717] Alternatively, the bioluminescence of luciferase-transduced AML cells was analyzed after 48 h of co-culture with CAR-T cells. Briefly, cells were centrifuged and resuspended in 50 μl PBS1X and then incubated with XenoLight D-luciferin firefly (15 mg / ml in PBS; Perkin Elmer, Waltham, MA) for 10 min. The cells were analyzed by Spark-Tecan multiwell plate reader (Tecan Group Ltd., Switzerland) measured luciferase activity and data signal reduction was reported as percentage of AML cell lysis assessed by bioluminescence imaging (BLI).

[0718] Colony formation assay

[0719] After 6 h of co-culture at a ratio of 1:1, a total of 2 × 10 3 Primary CD34 + Cells (isolated from healthy bone marrow or umbilical cord blood) were inoculated into 500 μl of MethoCult TM (H4534, Stemcell Technologies, MedaMB, Italy) and incubated at 37 ° C. For each co-culture, 4 copies were inoculated. After 14 days of inoculation, an appropriate volume of a 1:6 solution of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich–Merck) in Hanks' was added to the semi-solid medium. Images were obtained by optical microscopy with a camera, and colonies were counted using ImageJ software.

[0720] In vivo experiments in NSG mice

[0721] Procedures involving animals and their care were performed in accordance with institutional guidelines (EEC Council Directive 86 / 609, OJ L 358, 12 December 1987) and the “ARRIVE” guideline (Animals in Research Reporting In Vivo Experiments) in compliance with national and international laws and policies. Departmental authorization: 131 / 2022-PR. NSG mice (NOD.Cg-PrkdcscidII2rgtm1Wjl / SzJ, female, 4-5 weeks old, 20-25 g / mouse, maximum 5 animals / cage) were injected intravenously (tail vein) with 0.75 × 10 6 SHI-1-LUC (transduced with luciferase gene) cells. Two days after AML injection, 1.5 × 10 6 Mice were treated with CAR-T or untransduced / mock-transduced T cells as controls. Bioluminescence was monitored by intraperitoneal injection of XenoLight D-luciferin firefly (15 mg / ml in PBS; PerkinElmer) 10 minutes before measurement (Xenogen IVIS Spectral Bioluminescence / Optical Imaging System, Xenogen Corporation, Alameda, CA) to verify tumor growth.

[0722] Statistical analysis

[0723] Significant differences in means were detected by Student's t test or Wilcoxon nonparametric test according to the mean distribution. One-way multiple comparisons ANOVA with Tukey's multiple comparison test was used when more than 2 groups / conditions were compared. Graphs and related statistical analyses were generated using GraphPad Prism 8. All data are expressed as mean ± standard error of the mean (SEM). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 values ​​were statistically significant.

[0724] result

[0725] The data presented in this article confirm that:

[0726] In a large series of primary cases collected at diagnosis and relapse of myeloid and lymphoid acute leukemias, high expression of all six antigens was associated with very low / zero expression on healthy regenerating myeloid cells and hematopoietic stem cells; and

[0727] CAR-T cells created with the new ScFv that recognizes CD69 and CD84 showed specific lytic activity against AML blasts in vitro and in murine xenografts transplanted with AML cell lines.

[0728] The data support the development of CD63-, CD151-, CD72-, CD84-, CD69-, or CD109-based immunotherapeutic and diagnostic approaches for myeloid and lymphoid leukemias.

[0729] 4.4 Example 4: Validation of CAR-T binding and protocell lysis in zebrafish

[0730] To test the binding efficiency of scFv to TSA, 48 hours after fertilization, GFP expressing high levels of TSA was transfected into the ovum at a 1:1 ratio. + AML cell lines (and GFP not expressing TSA + Cell lines as control) and mCherry + A mixed solution of CAR-T or control T cells (hereinafter referred to as "mixed cells") was injected into zebrafish embryos.

[0731] In transplanted animals, the number of circulating fluorescent GFP / mCherry-positive cells and yellow fusion complexes representing bound complexes were assessed in all groups of animals (approximately 30 embryos per group) by fluorescence microscopy and ImageJ software at 1, 5, 24, and 72 hours after injection to determine the binding between AML and CAR T cells.

[0732] Next, the specificity and killing efficacy of the scFv were evaluated. The proliferation / death of xenografted CAR-T cells and the reduction of AML blasts in embryos were evaluated.

[0733] At 5 and 24 h after injection, 25 embryos / group were sacrificed to count GFP by flow cytometry analysis. + and mCherry + Comparison between reporter-expressing specific TSA-CAR T cells and control vector demonstrates the potency of lysis that occurs upon binding.

[0734] 4.5 Example 5: In vitro lytic efficacy of anti-CD84 (B8 and F12) and anti-CD69 (A1, F1, C2 and H2) CAR-T cells on primary AML cells

[0735] To test the activity of anti-CD84 CAR-T cells, CAR-T cells containing scFvB8 and F12 were cultured in culture medium alone (---) or co-cultured with AML cells from AML patient-derived xenograft models (AML-PDX).

[0736] Generation of AML-PDX

[0737] Xenografts (PDX) are generated using blasts derived from the bone marrow of pediatric patients diagnosed with de novo AML. Fig.29 Expression of TSA CD84, CD69, and CD72 in primary AML cells derived from AML-PDXs is shown. Specific CAR candidates from previous experiments were tested on AML-PDXs.

[0738] For PDX generation, NOD.Cg-Prkdc was transfected 24 hours prior to leukemic cell transplantation. scid Iq tm1Wjl / SzJ(NSG) mice (4-8 weeks old) were irradiated with 1.5 Gy. Briefly, cells previously depleted of CD3 were isolated by immuno-magnetic cell separation (Miltenyi Biotec) using the CD3 MicroBead Kit (Miltenyi Biotec). + T cells to prevent graft-versus-host disease (GV H D) 1×10 6 Primary AML cells were injected intravenously (iv) into first generation mouse recipients (P0). To monitor engraftment, human CD45 in peripheral blood (PB) was measured by flow cytometry every two weeks starting 1 month after AML cell injection. + (hCD45 + ) cells to assess tumor burden. hCD45 in PB + Cells > 5% indicate AML engraftment, in hCD45 + Mice were sacrificed when the number of cells was ≥20%. Organs (femur and spleen) were recovered and rinsed, and mechanically dissociated to harvest cells for biobanking. After two serial passages, 1×10 6 hCD45 + Cells were injected intravenously into secondary and tertiary recipient mice to generate P1- and P2-PDX models. P2 model stability was tested by RNA and exome sequencing. Ex vivo cells were used for in vitro testing.

[0739] When anti-CD84 CAR-T cells (B8 and F12) and AML cells from AML-PDX models were co-cultured, anti-CD84 CAR-T cells (B8 and F12) showed lytic efficacy against target cells, such as Fig.30The results are shown in the “% Killing” in the table. When normalized to the lytic potency of empty CAR T cells, the lytic potency of CAR-T cells (B8 and F12) was observed, confirming that the CAR expression cassette does not alter T cell function and that CD84 is a specific target in primary AML. Consistently, the in vitro antitumor activity of B8 and F12 anti-CD84 CAR-T cells ranged from 5 to 80% in AML cells from 5 different AML models. The persistence of activated CAR-T cells was monitored in vitro for up to 48 hours ( Fig.30 ).

[0740] To detect newly generated CD69 CAR-T cells, the activity of ScFvA1, F1, C2 and H2 against CD69 was tested in different CAR-T cell products. CAR-T cells were maintained in culture medium alone or co-cultured with target AML cell lines (SHI-1 and HL60 cell lines) expressing CD69 antigen. HL-60 and SHI-1 AML cell lines were used as target cells, and U937 and K562 cell lines were used as negative controls. These cell lines were grown in RPMI (Life Technologies; 11875093) supplemented with 10% fetal bovine serum (FBS; Life Technologies; 10270106), 1% penicillin-streptomycin (P / S, 10000U / mL, Life Technologies; 15140148) and 1% L-glutamine (200mM, ThermoFisher; 25030024), except for the SHI-1 cell line, which was cultured in DMEM (Life Technologies; 41965039) supplemented with 10% FBS and 1% P / S, and HEK293T was grown in IMDM (Euroclone; ECB2072L) supplemented with 10% FBS, 1% P / S and 1% L-glutamine. All cells were cultured at 37°C in a humidified incubator with 5% CO2. Primary AML ex vivo cells were cultured at 37°C in RPMI medium 1640 with 10% FBS, 2 mM glutamine (Gibco, Life Technologies), 100 U / mL streptomycin / penicillin (Gibco, Life Technologies), and supplemented with 50 ng / mL thrombopoietin (TPO), 50 ng / mL stem cell factor (SCF), 50 ng / mL FMS-like tyrosine kinase 3 ligand (Flt3L), 20 ng / mL interleukin (IL)-3, and 20 ng / mL IL-6, all purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

[0741] When co-cultured with a target AML cell line expressing the CD69 antigen, CD69 CAR-T cells showed lytic efficacy in SHI-1 and HL60 target cell lines (Figure 31). No results were observed on empty CAR T cells (MOI 5, simulated transduced T cells) that were identical to those of CD69 CAR-T cells. Consistently, significant in vitro antitumor activity against target cell lines was measured by the 4 ScFv sequences tested (A1, F1, C2, and H2 of Tables 13-14), ranging from 20 to 90%, depending on the cell line. Therefore, CAR-T products A1, F1, C2, and H2 showed desired efficacy and specificity in vitro monitoring for up to 48 hours.

[0742] The CAR-T cell lysis titer is calculated as follows:

[0743]

[0744] Each column in Figure 31 represents the killing percentage of AML samples by different CAR-T products.

[0745] 4.6 Example 6: In vivo efficacy of B8 and F12 CD84 ScFv and H3 ScFv against CD69 CAR-T cells

[0746] Two antigens, CD84 and CD69, were detected as targets for in vivo AML immunotherapy. CAR-T cells were prepared as described in Example 3, and four different specific ScFvs were identified in the human natural antigen binding fragment (Fab) phage library, namely F12 and B8 that recognize CD84, and H3, G1, A1, F1, C2 and H2 that recognize CD69. The nucleotide sequences are shown in Figure 27. The SHI-1 AML cell line expressing high levels of CD84 and CD69 was used as the target cell, as well as CD84 negative (CD84 - )'s U937( Fig.28A ) and K562( Fig.28B ) cell lines were used as non-target cells to detect the in vivo efficacy of the prepared CAR-T cells against CD84 (F12 and B8 chains) and CD69 (H3 chain) as well as the specificity of the prepared anti-CD84 B8 chain. + CD69 + )AML cell lines were engineered to express luciferase for non-invasive bioluminescent imaging (BLI). The prepared CAR-T cells targeting CD84 (B8 and F12 chains) and CD69 (H3 chain) were infused into NOD / SCIDγ (NSG) mice transplanted with target AML cell lines (for CD84, N=7, for CD69, N=10). Briefly, mice were injected with 0.5×10 6AML cells / mouse (day 1), and then (day 3) 1.5×10 6 CAR-T cells or mock-transduced T cells (MOI 5, empty CAR: mice).

[0747] Lysis activity

[0748] The lytic activities of CAR-T cells targeting CD84 and CAR-T cells targeting CD69 are shown in Fig.28A and Fig.27B As shown in the figure, mice injected with CAR-T cells showed expression of SHI-1 (CD84 + CD69 + ) significantly increased the lytic activity of AML cell lines, reducing leukemic burden, as measured by a decrease in bioluminescence (BLI) from day 24 to day 38 with B8 targeting CD84 (*p<0.05, **p<0.005, Mann Whitney test), from day 38 to day 45 with F12 targeting CD84 (*p<0.05, Mann Whitney test), and from day 24 to day 45 with H3 targeting CD69 (**p<0.005, ***p<0.0005, ****p<0.0001, Mann Whitney test). Figures 27A-27B As shown, the increase in lytic potency in CAR-T cell-injected mice is represented by a decrease in the luciferase bioluminescent signal, which is expressed as total flux. In contrast, in empty CAR-T cells, the luciferase signal increased from day 24 to day 45.

[0749] like Figures 27C-27D As shown, mice survived up to 90 days after injection of CAR-T cells containing scFvs of F12, B8 targeting CD84, and H3 targeting CD69, indicating that CAR T cell constructs containing F12, B8, or H3 scFv have additive in vivo efficacy in the treatment of AML ( Figures 27C-27D , *p<0.05Mantel-Cox test). In contrast, mice injected with empty CAR-T cells survived for approximately 49 days.

[0750] 4.7 Example 7: Specificity of B8CD84 scFvCAR-T cells in vivo

[0751] To test the specificity of the novel anti-CD84 B8 chain, anti-CD84 B8 CAR-T cells were infused into the U937 (CD84 neg ) and K562(CD84 neg )AML cell line-transplanted NSG mice were used as non-target cells lacking CD84 expression. neg) and K562(CD84 neg )AML cell line was engineered to express luciferase for non-invasive bioluminescence imaging (BLI). The prepared CD84-targeted CAR-T cells were infused into two U937 (CD84 neg ) and K562(CD84 neg ) AML non-target cell line transplanted NSG mice. Briefly, mice were injected with 0.5×10 6 AML cells / mouse (day 1), and then (day 3) 1.5×10 6 CAR-T cells or mock-transduced T cells (MOI 5, mCherry: mouse) (N = 6-10 animals / group). Figures 28A-28B As shown, mice injected with anti-CD84 CAR-T cells showed an increase in AML engraftment similar to that of empty CAR-T cells and no reduction in leukemic burden monitored by BLI until day 30 and day 22 after challenge with K562 and U937 cells, respectively. At the time of death or sacrifice of mice, the presence of T cells was monitored by CD3 as well as CD84 expression in the bone marrow and AML-infiltrated spleen. CD84 expression was not detected and the percentage of T cells ranged from 2 to 86%.

[0752] Three new ScFvs were found to be highly specific for the target AML cell line (one in Table 12 was specific for CD69 (H3), and two were specific for C84 (B8 and F12)), and showed high efficacy in NGS mice. In addition, engineered CAR-T cells containing ScFvs for CD69 (A1, F1, C2 or H2 in Table 12) were found to exert lytic efficacy against target cell lines in vitro.

[0753] Five AML patient-derived xenograft (PDX) models were established for their high expression of three TSAs (CD84, CD69, and CD72), as Fig.29 As shown, and when expressed by CAR T cells, it is targeted by the ScFv disclosed herein. PDX closely reproduces patient disease, meeting the specific needs of late-stage in vivo studies. PDX models are fundamental preclinical tools that effectively bridge laboratory and clinical data in translational research. The generation of PDX includes mimicking tumors by transferring the original sample directly into immunocompromised mice, avoiding intermediate in vitro culture passages that may induce genetic transformation or clonal selection. Therefore, the latest findings of TSA expression in AML-PDX confirm and support the findings in AML cell lines.

[0754] In vitro cytotoxicity assay

[0755] At the end of T cell expansion (day 17), cytotoxicity was determined by co-culturing AML target cell lines (antigen positive) or ex vivo AML with CAR-T cells or untransduced T cells at an E:T ratio of 1:1 for 48 hours. The co-cultured cells were stained with Annexin V-PE (Miltenyi Biotec; 130-118-363) and 7-AAD Staining Solution (Miltenyi Biotec; 130-11-568) and analyzed by flow cytometry using FACSCelestaTM CellAnalyzer and FlowJo software. The percentage of cell lysis was calculated using the following formula:

[0756]

[0757] In vivo experiments in NSG mice

[0758] Procedures involving animals and their care were performed in accordance with institutional guidelines (EEC Council Directive 86 / 609, OJ L 358, December 12, 1987) and the “ARRIVE” guideline (Animals in Research Reporting In Vivo Experiments) in accordance with national and international laws and policies. Departmental authorization: 131 / 2022-PR. NSG mice (4-5 weeks old, female, 20-25 g / mouse, maximum N = 5 animals / cage) were injected with 0.5×10 6 SHI-1-LUC (transduced with luciferase gene) cells. Two days after AML injection, 1.5 × 10 6 Mice were treated with CAR-T or untransduced / mock-transduced T cells as controls. Bioluminescence was monitored by intraperitoneal injection of XenoLight D-luciferin firefly (15 mg / ml in PBS; Perkin Elmer, Waltham, MA) 10 minutes before measurement (Xenogen IVIS Spectral Bioluminescence / Optical Imaging System, Xenogen Corporation, Alameda, CA) to verify tumor growth.

[0759] Statistical analysis

[0760] Graphs and associated statistical analyses were generated using Graphpad Prism 8. All data are presented as mean ± standard error of the mean (SEM). Values ​​are statistically significant.

[0761] Significant differences in means were detected by Mann Whitney test or Mantel-Cox test depending on the mean distribution. Graphs and related statistical analyses were generated using GraphPad Prism 8. All data are expressed as mean ± standard error of the mean (SEM). *p<0.05, **p<0.005, ***p<0.0005 and ****p<0.0001 values ​​were statistically significant.

[0762] 4.8 Example 8: Cytokine production capacity of new B8 and F12 CD84 ScFvCAR-T cells in vitro

[0763] Anti-CD84 CAR-T cells were prepared with two different ScFvs (i.e., F12 and B8) and tested for their ability to respond to SHI-1 and HL60 (expressing CD84) in vitro. + and CD69 + ) The ability of the target antigen on the AML cell line to produce T cell-related cytokines.

[0764] HL-60 and SHI-1 AML cell lines used as target cells were grown in RPMI and DMEM (Life Technologies; 41965039; 11875093), respectively, supplemented with 10% fetal bovine serum (FBS; Life Technologies; 10270106), 1% penicillin-streptomycin (P / S, 10000 U / mL, Life Technologies; 15140148), and 1% L-glutamine (200 mM, ThermoFisher; 25030024). SHI-1-CD84 koAML cell line is used as the control (Synthego Corporation, Redwood City, CA) of knocking out CD84 gene.All cells are cultured in a humidified incubator with 5% CO2.Primary AML ex vivo cells are cultured in RPMI culture medium 1640 at 37°C, and the RPMI culture medium 1640 has 10% FBS, 2mM glutamine (Gibco, Life Technologies), 100U / mL streptomycin / penicillin (Gibco, Life Technologies), and is supplemented with 50ng / mL thrombopoietin (TPO), 50ng / mL stem cell factor (SCF), 50ng / mL FMS-like tyrosine kinase 3 ligands (Flt3L), 20ng / mL interleukin-3 (IL-3) and 20ng / mL interleukin-6 (IL-6), all cytokines are purchased from Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, DE).

[0765] B8 or F12 CAR T cells or CAR T cells with empty vector were cultured with target AML cell lines SHI-1 or HL6 at an effector to target (E:T) ratio of 1: 1 for 48 hours. The cells were then labeled for cell surface antigen expression, fixed, permeabilized and stained with anti-IFNγ and anti-TNFα antibodies and analyzed by flow cytometry to determine the percentage of cells expressing IFNγ or anti-TNFα.

[0766] Cytokine intracellular staining

[0767] After co-culture of B8 CD84 or F12 CD84 CAR-T cells with AML cells SHI-1 or HL-60 (1:1E:T ratio), and A1 CD69 or C2 CD69 CAR T cells with AML cells SHI-1 or HL-60 (1:1E:T ratio) for 6, 24, and 48 hours, the cytokine production capacity of B8 CD84, F12 CD84, A1 CD69, and C2 CD69 CAR-T cells was evaluated by flow cytometry. Briefly, to distinguish effector CAR T cells from target AML cells, 0.4 × 10 6Cells were initially labeled for cell surface antigen expression (i.e., CD45, CD3, CD33). Then, cells were fixed, permeabilized, and stained with anti-IFNγ and anti-TNFα antibodies using the Inside Stain Kit (Miltenyi Biotec; 130-090-477) according to the manufacturer's instructions. Finally, the percentage of IFNγ and TNFα positive expressing cells was estimated relative to total lymphocytes, and data were presented as histograms with replicate mean ± SEM.

[0768] result

[0769] When combined with the target SHI-1 and HL60 (CD84 + / CD69 + ) AML cell lines, CAR-T cells prepared with B8 and F12 scFV chains targeting CD84 (interchangeably used herein as "B8 CD84" and "F12 CD84" CAR-T cells) showed a higher frequency of positive cells expressing IFNγ and TNFα (respectively) compared to CAR-T cells alone ("---"). Fig.32A and Fig.32B ), with an effector:target (E:T) ratio of 1:1, which confirmed their ability to appropriately produce inflammatory cytokines after antigen binding (*p<0.05, **p<0.005, Mann Whitney T-test, n=1-4 independent experiments). 4.9 Example 9: Anti-CD84 CAR-T cells against CD34 + Hematopoietic stem and progenitor cells (HSPC) show no toxicity

[0770] In a standard colony forming unit (CFU) assay, the effector:target ratio (E:T) was 1:1 against commercially available healthy donor CD34 + Hematopoietic stem / progenitor cells (HSC) were used to study the in vitro inactivation of anti-CD84 CAR-T cells.

[0771] CFU assay

[0772] Commercially available CD34 from healthy donors +Cells (Hu BM CD34+, StemExpress; BM34001C) were co-cultured with medium alone, empty CAR, B8 CD84 or F12CD84 CAR T cells at a 1:1 effector: target ratio for 4 hours. After incubation, the cell suspension was added to a semi-solid methylcellulose-based medium Methocult H4534Classic (StemCell Technologies Inc, Vancouver, British Columbia, Canada) without EPO and seeded into a 3 cm tissue culture dish. After 10-12 days, colonies derived from granulocyte-macrophages (G, M and GM) and multipotent granulocytes, erythrocytes, macrophages, megakaryocytes (GEMM) progenitor cells were scored and counted according to the manufacturer's instructions, and the total number of colonies was evaluated. Data are expressed as the mean ± SEM of independent replicates.

[0773] result

[0774] B8 CD84 and F12 CD84 CAR T cells, as well as constructs made with B8 and F12 scFv chains specific for the CD84 antigen, are specific for CD34 + The cells did not produce significant effects, such as Figures 33A-33B B8CD84 and F12CD84CAR T cells do not induce CD34 + The decrease in the total number of CFU formed by HSC ( Fig.33A ), nor does it induce CD34 + Reduction in HSC colony subtypes (granulocyte-macrophage (G, M, and GM) and multipotent granulocyte, erythroid, macrophage, megakaryocyte (GEMM) progenitors) ( Fig.33B ). This result supports the finding that all hematopoietic progeny can reconstitute ( Fig.33B ).

[0775] 4.10 Example 10: In vivo specificity of novel B8CD84ScFvCAR-T cells

[0776] To test the specificity of B8CD84 and F12CD84 CAR-T cells, SHI-1-CD84 ko AML cell lines were used as non-target cell lines without CD84 expression. Specifically, this luciferase-transduced AML cell line was third-party engineered and purchased from Synthego Corporation to genetically knock out the CD84 antigen, as confirmed by flow cytometry analysis. The prepared B8 and F12 CD84 CAR-T cells targeting CD84 were infused into the SHI-1-CD84 expressing luciferase.ko AMLL cell line was transplanted into NOD / SCIDγ (NSG) mice.

[0777] Procedures involving animals and their care were performed in accordance with institutional guidelines (EEC Council Directive 86 / 609, OJ L 358, 12 December 1987) and the “ARRIVE” guideline (Animals in Research Reporting In Vivo Experiments) in compliance with national and international laws and policies. Departmental authorization: 131 / 2022-PR. NSG mice (NOD.Cg-PrkdcscidII2rgtm1Wjl / SzJ, female, 4-5 weeks old, 20-25 g / mouse, maximum 5 animals / cage) were injected intravenously (tail vein) with 0.5 × 10 6 SHI-1-LUC (transduced with luciferase gene) cells or HL60-LUC cells. Two days after AML injection (day 3), 1.5 × 10 6 Mice were treated with target or mock-transduced T cells (empty CAR, multiplicity of infection (MOI) 5mCherry / mouse) as controls.

[0778] result

[0779] B8 and F12 CD84 CAR-T cell-injected mice showed an increase in AML engraftment comparable to that of empty CAR-infused mice, with no reduction in leukemic burden monitored by bioluminescence imaging (BLI) from day 15 to day 45 ( Fig.34A The survival rates of B8CD84 CAR-T cell-infused mice and F12CD84 CAR-T cell-infused mice were also comparable to that of mice injected with empty CAR ( Fig.34B ).

[0780] 4.11 Example 11: In vitro lytic efficacy of B8 CD84 and F12 CD84 CAR-T cells and A1 CD69 and C2 CD69 CAR-T cells on primary childhood AML cells isolated from vitro

[0781] To test the activity of the newly generated CAR-T cells targeting CD84 (B8 or F12 CD84 CAR-T cells) and CD69 (A1 or C2 CD69 CAR-T cells), CD84 and CD69 CAR-T cells were maintained in separate culture media or co-cultured with AML cells from AML patient-derived xenograft models (AML-PDX).

[0782] Generation of AML-PDX

[0783] For PDX generation, NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice (4-8 weeks old) were irradiated with 1.5 Gy 24 hours before leukemic cell transplantation. Briefly, cells previously depleted of CD3 were isolated by immuno-magnetic cell separation (Miltenyi Biotec) using the CD3 MicroBead Kit (Miltenyi Biotec). + T cells to prevent graft-versus-host disease (GV H D) 1×10 6 Primary AML cells were injected intravenously (iv) into first generation mouse recipients (P0). To monitor engraftment, tumor burden was assessed by measuring hCD45 positive cells in peripheral blood (PB) by flow cytometry every two weeks starting 1 month after AML cell injection. hCD45 cells >5% in PB indicated AML engraftment, and mice were sacrificed at 20%. Organs (femurs and spleens) were recovered and flushed or mechanically dissociated for cell harvest and biobanking. For two serial passages, 1×10 6 Human CD45 (hCD45) was injected intravenously into secondary and tertiary recipient mice to generate P1- and P2-PDX models. P2 model stability was tested by RNA and exome sequencing. Ex vivo cells were used for in vitro testing; the P2 generation was expanded for in vivo testing.

[0784] Cytotoxicity assay

[0785] At the end of T cell expansion (day 17), cytotoxicity was determined by co-culturing ex vivo PDX-AML cells with CAR-T cells or empty CAR-T cells at an E:T ratio of 1:1 for 48 hours. The co-cultured cells were stained with Annexin V-PE (Miltenyi Biotec; 130-118-363) and 7-AAD Staining Solution (Miltenyi Biotec; 130-11-568) and analyzed by flow cytometry using FACSCelestaTM Cell Analyzer and FlowJo software. The percentage of killing was calculated using the following formula:

[0786]

[0787] result

[0788] B8 and F12 CD84 ( Fig.35A ) and A1 and C2 CD69 ( Fig.35B)CAR-T cells show high lysis efficacy on primary target cells derived from 3 different pediatric AML-PDX models, as shown in the percentage of killing of AML-PDX cells after co-culture. In three different AML cases (PDX#3, 6 and 7), the lysis efficacy was 5-70%. Each column represents the percentage of killing of AML samples by different CAR-T products.

[0789] The results showed that B8 and F12 CD84 CAR-T cells and A1 and C2 CD69 CAR-T cells were effective against all PDX-derived AML cells ( Figures 35A-35B The positive and consistent anti-tumor activity of PDX#3, PDX#6 and PDX#7) was observed in the samples.

[0790] 4.12 Example 12: Cytokine production ability of B8 and F12 CD84 CAR-T cells on AML-PDX cells in vitro

[0791] The ability of B8 and F12 CD84 CAR-T cells to produce T cell-related cytokine function in response to binding to primary AML cells derived from AML-PDXs was tested in vitro. Briefly, B8 and F12 CD84 CAR T or empty CAR cells were cultured alone (---) or at a 1:1 E:T ratio with target cells derived from three different PDX-AML models for 48 hours, followed by labeling cells for cell surface antigen expression, fixation, permeabilization and staining with anti-IFNγ and anti-TNFα antibodies as described in Example 8 and analysis by flow cytometry.

[0792] result

[0793] When compared with AML-PDX target cells ( Figures 36A-36B When cultured with AML-PDXs (PDX#3, PDX#6, and PDX#7), B8 and F12 CD84 CAR T cells targeting CD84 showed a higher percentage of cells positive for IFNγ and TNFα (P<0.05, PDX#4, PDX#5, and PDX#6, respectively) compared to when AML-PDXs were cultured with empty CAR T cells. Fig.36A and 36B ), which confirmed the enhanced ability to appropriately produce inflammatory cytokines upon antigen binding (*p<0.05, **p<0.005, Mann Whitney T-test, n=1-4 independent experiments).

[0794] 4.13 Example 13: In vivo anti-tumor lytic activity of new B8CD84ScFvCAR-T cells against AML-PDX

[0795] To test the potency and specificity of the newly generated B8CD84 CAR T cells in vivo, an AML-PDX model obtained from a pediatric patient diagnosed with AML expressing high levels of CD84 was used.

[0796] 10 cells collected from AML-PDX models transduced with the luciferase gene were used. 6 10×10 AML ex vivo cells were intravenously injected into NSG mice. Then, on day 28+ after AML cell injection, 5 or 10×10 6 CAR-T cells were tail-injected into mice. Bioluminescence was monitored to verify tumor growth by intraperitoneal injection of XenoLight D-luciferin firefly (15 mg / ml in PBS; Perkin Elmer) 10 minutes before measurement (Xenogen IVIS Spectral Bioluminescence / Optical Imaging System, Xenogen Corporation, Alameda, CA).

[0797] Briefly, NSG mice were injected with 6 10 cells expressing AML-luciferase (LUC), and AML engraftment and spread were monitored weekly by LUC bioluminescence (expressed as total flux). On day +28 of AML injection, LUC analysis produced a reliable signal of AML engraftment, and mice were then tail-injected with 5×10 6 B8 CD84 CAR-T cells (E:T 5:1 ratio) or 10×10 6 B8CD84 CAR-T cells (E:T 10:1 ratio, n = 5-10 animals / group, Fig.37A ). Disease progression was monitored at day +7 after CAR-T infusion, independent of the effector-to-target ratio they were infused with ( Fig.37B ), showing that both CAR-T cells and AML cells were present in the peripheral blood (PB), bone marrow (BM), and spleen (SPL) of empty and B8 CAR T-vaccinated mice, as shown in Fig.37B As shown in “% of lymphocytes” on day +7. When CAR T cells encountered AML cells, the CAR-T cells expanded as indicated by the increase in lymphocytes present in PB, BM, and SPL in the empty CAR and B8CD84CAR-T cell groups at E:T 5:1 and E:T 10:1 ratios and the absence of AML cells, although lower amounts of AML cells were present in PB in both groups.

[0798] like Fig.37C As shown, on day +12 after CAR-T cell infusion, both B8CD84CAR T cells and empty CAR cells were present in BM and SPL, but not in PB, as shown in Fig.37CThe "% of lymphocytes" is shown in Figure 1. Fig.37C As shown in the “% of AML cells”, AML cells were detected only in PB and BM in mice administered with empty CAR. A small number of AML cells were detected in the SPL of mice administered with B8CD84CAR.

[0799] like Fig.37C As shown, on day +16 after CAR-T cell infusion, both B8CD84CAR T cells and empty CAR cells were present in PB, BM, and SPL, as shown in Fig.37C In addition, mice infused with B8CD84CAR-T cells showed total AML clearance in all organs (PB, BM, and SPL) on day +16. AML cells were only present in nullCAR T cells in PB, BM, and SPL.

[0800] Together, these results suggest that B8CD84 CAR T cells effectively eliminate primary AML cells and rapidly reduce disease burden.

[0801] Next, use 10 6 Different doses (5 × 10 6 cells or 10×10 6 The effectiveness of B8 CD84 CAR T cells ( Fig.38A Flow cytometric analysis of cell distributi...

Claims

1. An antigen binding protein (ABP) that specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

2. The ABP according to claim 1, which specifically binds to human CD84.

3. The ABP of claim 2, wherein the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:97, SEQ ID NO:98 or SEQ ID NO:

90.

4. The ABP of claim 3, wherein the ABP comprises: a. V having the sequence of SEQ ID NO: 131 L CDR1, V having the sequence of SEQ ID NO: 132 L CDR2, V having the sequence of SEQ ID NO: 133 L CDR3, V having the sequence of SEQ ID NO: 251 H CDR1, V having the sequence of SEQ ID NO: 252 H CDR2 and V having the sequence of SEQ ID NO: 253 H CDR3; b. V having the sequence of SEQ ID NO: 134 L CDR1, V having the sequence of SEQ ID NO: 135 L CDR2, V having the sequence of SEQ ID NO: 136 L CDR3, V having the sequence of SEQ ID NO: 254 H CDR1, V having the sequence of SEQ ID NO: 255 H CDR2 and V having the sequence of SEQ ID NO: 256 H CDR3; c. V having the sequence of SEQ ID NO: 110 L CDR1, V having the sequence of SEQ ID NO: 111 L CDR2, V having the sequence of SEQ ID NO: 112 L CDR3, V having the sequence of SEQ ID NO: 230 H CDR1, V having the sequence of SEQ ID NO: 231 H CDR2 and V having the sequence of SEQ ID NO: 232 H CDR3; d. V having the sequence of SEQ ID NO: 161 L CDR1, V having the sequence of SEQ ID NO: 162 L CDR2, V having the sequence of SEQ ID NO: 163 L CDR3, V having the sequence of SEQ ID NO: 281 H CDR1, V having the sequence of SEQ ID NO: 282 H CDR2 and V having the sequence of SEQ ID NO: 283 H CDR3; e. V having the sequence of SEQ ID NO: 164 L CDR1, V having the sequence of SEQ ID NO: 165 L CDR2, V having the sequence of SEQ ID NO: 166 L CDR3, V having the sequence of SEQ ID NO: 284 H CDR1, V having the sequence of SEQ ID NO: 285 H CDR2 and V having the sequence of SEQ ID NO: 286 H CDR3; f. V having the sequence of SEQ ID NO: 140 L CDR1, V having the sequence of SEQ ID NO: 141 L CDR2, V having the sequence of SEQ ID NO: 142 L CDR3, V having the sequence of SEQ ID NO: 260 H CDR1, V having the sequence of SEQ ID NO: 261 H CDR2 and V having the sequence of SEQ ID NO: 262 H CDR3; g. V having the sequence of SEQ ID NO: 191 L CDR1, V having the sequence of SEQ ID NO: 192 L CDR2, V having the sequence of SEQ ID NO: 193 L CDR3, V having the sequence of SEQ ID NO: 311 H CDR1, V having the sequence of SEQ ID NO: 312 H CDR2 and V having the sequence of SEQ ID NO: 313 H CDR3; h. V having the sequence of SEQ ID NO: 194 L CDR1, V having the sequence of SEQ ID NO: 195 L CDR2, V having the sequence of SEQ ID NO: 196 L CDR3, V having the sequence of SEQ ID NO: 314 H CDR1, V having the sequence of SEQ ID NO: 315 H CDR2 and V having the sequence of SEQ ID NO: 316 H CDR3; i. V having the sequence of SEQ ID NO: 170 L CDR1, V having the sequence of SEQ ID NO: 171 L CDR2, V having the sequence of SEQ ID NO: 172 L CDR3, V having the sequence of SEQ ID NO: 290 H CDR1, V having the sequence of SEQ ID NO: 291 H CDR2 and V having the sequence of SEQ ID NO: 292 H CDR3; j. V having the sequence of SEQ ID NO: 221 L CDR1, V having the sequence of SEQ ID NO: 222 L CDR2, V having the sequence of SEQ ID NO: 223 L CDR3, V having the sequence of SEQ ID NO: 341 H CDR1, V having the sequence of SEQ ID NO: 342 H CDR2 and V having the sequence of SEQ ID NO: 343 H CDR3; or V having the sequence of SEQ ID NO: 224 L CDR1, V having the sequence of SEQ ID NO: 225 L CDR2, V having the sequence of SEQ ID NO: 226 L CDR3, V having the sequence of SEQ ID NO: 344 H CDR1, V having the sequence of SEQ ID NO: 345 H CDR2 and V having the sequence of SEQ ID NO: 346 H CDR3; or k. V having the sequence of SEQ ID NO: 200 L CDR1, V having the sequence of SEQ ID NO: 201 L CDR2, V having the sequence of SEQ ID NO: 202 L CDR3, V having the sequence of SEQ ID NO: 320 H CDR1, V having the sequence of SEQ ID NO: 321 H CDR2 and V having the sequence of SEQ ID NO: 322 H CDR3.

5. The ABP according to any one of claims 1 to 4, comprising: a. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 80, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 88; or b. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:81, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:88; or c. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

83. The ABP according to claim 1 , which specifically binds to human CD69.

7. The ABP of claim 6, wherein the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 89-96.

8. The ABP according to any one of claims 6 to 7, wherein the ABP comprises: a. V having the sequence of SEQ ID NO: 107 L CDR1, V having the sequence of SEQ ID NO: 108 L CDR2, V having the sequence of SEQ ID NO: 109 L CDR3, V having the sequence of SEQ ID NO: 227 H CDR1, V having the sequence of SEQ ID NO: 228 H CDR2 and V having the sequence of SEQ ID NO: 229 H CDR3; b. V having the sequence of SEQ ID NO: 110 L CDR1, V having the sequence of SEQ ID NO: 111 L CDR2, V having the sequence of SEQ ID NO: 112 L CDR3, V having the sequence of SEQ ID NO: 230 H CDR1, V having the sequence of SEQ ID NO: 231 H CDR2 and V having the sequence of SEQ ID NO: 232 H CDR3; c. V having the sequence of SEQ ID NO: 113 L CDR1, V having the sequence of SEQ ID NO: 114 L CDR2, V having the sequence of SEQ ID NO: 115 L CDR3, V having the sequence of SEQ ID NO: 233 H CDR1, V having the sequence of SEQ ID NO: 234 H CDR2 and V having the sequence of SEQ ID NO: 235 H CDR3; d. V having the sequence of SEQ ID NO: 116 L CDR1, V having the sequence of SEQ ID NO: 117 L CDR2, V having the sequence of SEQ ID NO: 118 L CDR3, V having the sequence of SEQ ID NO: 236 H CDR1, V having the sequence of SEQ ID NO: 237 H CDR2 and V having the sequence of SEQ ID NO: 238 H CDR3; e. V having the sequence of SEQ ID NO: 119 L CDR1, V having the sequence of SEQ ID NO: 120 L CDR2, V having the sequence of SEQ ID NO: 121 L CDR3, V having the sequence of SEQ ID NO: 239 H CDR1, V having the sequence of SEQ ID NO: 240 H CDR2 and V having the sequence of SEQ ID NO: 241 H CDR3; f. V having the sequence of SEQ ID NO: 122 L CDR1, V having the sequence of SEQ ID NO: 123 L CDR2, V having the sequence of SEQ ID NO: 124 L CDR3, V having the sequence of SEQ ID NO: 242 H CDR1, V having the sequence of SEQ ID NO: 243 H CDR2 and V having the sequence of SEQ ID NO: 244 H CDR3; g. V having the sequence of SEQ ID NO: 125 L CDR1, V having the sequence of SEQ ID NO: 126 L CDR2, V having the sequence of SEQ ID NO: 127 L CDR3, V having the sequence of SEQ ID NO: 245 H CDR1, V having the sequence of SEQ ID NO: 246 H CDR2 and V having the sequence of SEQ ID NO: 247 H CDR3; h. V having the sequence of SEQ ID NO: 128 L CDR1, V having the sequence of SEQ ID NO: 129 L CDR2, V having the sequence of SEQ ID NO: 130 L CDR3, V having the sequence of SEQ ID NO: 248 H CDR1, V having the sequence of SEQ ID NO: 249 H CDR2 and V having the sequence of SEQ ID NO: 250 H CDR3; i. V having the sequence of SEQ ID NO: 137 L CDR1, V having the sequence of SEQ ID NO: 138 L CDR2, V having the sequence of SEQ ID NO: 139 L CDR3, V having the sequence of SEQ ID NO: 257 H CDR1, V having the sequence of SEQ ID NO: 258 H CDR2 and V having the sequence of SEQ ID NO: 259 H CDR3; j. V having the sequence of SEQ ID NO: 140 L CDR1, V having the sequence of SEQ ID NO: 141 L CDR2, V having the sequence of SEQ ID NO: 142 L CDR3, V having the sequence of SEQ ID NO: 260 H CDR1, V having the sequence of SEQ ID NO: 261 H CDR2 and V having the sequence of SEQ ID NO: 262 H CDR3; k. V having the sequence of SEQ ID NO: 143 L CDR1, V having the sequence of SEQ ID NO: 144 L CDR2, V having the sequence of SEQ ID NO: 145 L CDR3, V having the sequence of SEQ ID NO: 263 H CDR1, V having the sequence of SEQ ID NO: 264 H CDR2 and V having the sequence of SEQ ID NO: 265 H CDR3; 1. V having the sequence of SEQ ID NO: 146 L CDR1, V having the sequence of SEQ ID NO: 147 L CDR2, V having the sequence of SEQ ID NO: 148 L CDR3, V having the sequence of SEQ ID NO: 266 H CDR1, V having the sequence of SEQ ID NO: 267 H CDR2 and V having the sequence of SEQ ID NO: 268 H CDR3; m. V having the sequence of SEQ ID NO: 149 L CDR1, V having the sequence of SEQ ID NO: 150 L CDR2, V having the sequence of SEQ ID NO: 151 L CDR3, V having the sequence of SEQ ID NO: 269 H CDR1, V having the sequence of SEQ ID NO: 270 H CDR2 and V having the sequence of SEQ ID NO: 271 H CDR3; n. V having the sequence of SEQ ID NO: 152 L CDR1, V having the sequence of SEQ ID NO: 153 L CDR2, V having the sequence of SEQ ID NO: 154 L CDR3, V having the sequence of SEQ ID NO: 272 H CDR1, V having the sequence of SEQ ID NO: 273 H CDR2 and V having the sequence of SEQ ID NO: 274 H CDR3; o. V having the sequence of SEQ ID NO: 155 L CDR1, V having the sequence of SEQ ID NO: 156 L CDR2, V having the sequence of SEQ ID NO: 157 L CDR3, V having the sequence of SEQ ID NO: 275 H CDR1, V having the sequence of SEQ ID NO: 276 H CDR2 and V having the sequence of SEQ ID NO: 277 H CDR3; p. V having the sequence of SEQ ID NO: 158 L CDR1, V having the sequence of SEQ ID NO: 159 L CDR2, V having the sequence of SEQ ID NO: 160 L CDR3, V having the sequence of SEQ ID NO: 278 H CDR1, V having the sequence of SEQ ID NO: 279 H CDR2 and V having the sequence of SEQ ID NO: 280 H CDR3; q. V having the sequence of SEQ ID NO: 167 L CDR1, V having the sequence of SEQ ID NO: 168 L CDR2, V having the sequence of SEQ ID NO: 169 L CDR3, V having the sequence of SEQ ID NO: 287 H CDR1, V having the sequence of SEQ ID NO: 288 H CDR2 and V having the sequence of SEQ ID NO: 289 H CDR3; r. V having the sequence of SEQ ID NO: 170 L CDR1, V having the sequence of SEQ ID NO: 171 L CDR2, V having the sequence of SEQ ID NO: 172 L CDR3, V having the sequence of SEQ ID NO: 290 H CDR1, V having the sequence of SEQ ID NO: 291 H CDR2 and V having the sequence of SEQ ID NO: 292 H CDR3; s. V having the sequence of SEQ ID NO: 173 L CDR1, V having the sequence of SEQ ID NO: 174 L CDR2, V having the sequence of SEQ ID NO: 175 L CDR3, V having the sequence of SEQ ID NO: 293 H CDR1, V having the sequence of SEQ ID NO: 294 H CDR2 and V having the sequence of SEQ ID NO: 295 H CDR3; t. V having the sequence of SEQ ID NO: 176 L CDR1, V having the sequence of SEQ ID NO: 177 L CDR2, V having the sequence of SEQ ID NO: 178 L CDR3, V having the sequence of SEQ ID NO: 296 H CDR1, V having the sequence of SEQ ID NO: 297 H CDR2 and V having the sequence of SEQ ID NO: 298 H CDR3; u. V having the sequence of SEQ ID NO: 179 L CDR1, V having the sequence of SEQ ID NO: 180 L CDR2, V having the sequence of SEQ ID NO: 181 L CDR3, V having the sequence of SEQ ID NO: 299 H CDR1, V having the sequence of SEQ ID NO: 300 H CDR2 and V having the sequence of SEQ ID NO: 301 H CDR3; v. V having the sequence of SEQ ID NO: 182 L CDR1, V having the sequence of SEQ ID NO: 183 L CDR2, V having the sequence of SEQ ID NO: 184 L CDR3, V having the sequence of SEQ ID NO: 302 H CDR1, V having the sequence of SEQ ID NO: 303 H CDR2 and V having the sequence of SEQ ID NO: 304 H CDR3; w. V having the sequence of SEQ ID NO: 185 L CDR1, V having the sequence of SEQ ID NO: 186 L CDR2, V having the sequence of SEQ ID NO: 187 L CDR3, V having the sequence of SEQ ID NO: 305 H CDR1, V having the sequence of SEQ ID NO: 306 H CDR2 and V having the sequence of SEQ ID NO: 307 H CDR3; x. V having the sequence of SEQ ID NO: 188 L CDR1, V having the sequence of SEQ ID NO: 189 L CDR2, V having the sequence of SEQ ID NO: 190 L CDR3, V having the sequence of SEQ ID NO: 308 H CDR1, V having the sequence of SEQ ID NO: 309 H CDR2 and V having the sequence of SEQ ID NO: 310 H CDR3; y. V having the sequence of SEQ ID NO: 197 L CDR1, V having the sequence of SEQ ID NO: 198 L CDR2, V having the sequence of SEQ ID NO: 199 L CDR3, V having the sequence of SEQ ID NO: 317 H CDR1, V having the sequence of SEQ ID NO: 318 H CDR2 and V having the sequence of SEQ ID NO: 319 H CDR3; z. V having the sequence of SEQ ID NO: 200 L CDR1, V having the sequence of SEQ ID NO: 201 L CDR2, V having the sequence of SEQ ID NO: 202 L CDR3, V having the sequence of SEQ ID NO: 320 H CDR1, V having the sequence of SEQ ID NO: 321 H CDR2 and V having the sequence of SEQ ID NO: 322 H CDR3; aa. V having a sequence of SEQ ID NO: 203 L CDR1, V having the sequence of SEQ ID NO: 204 L CDR2, V having the sequence of SEQ ID NO: 205 L CDR3, V having the sequence of SEQ ID NO: 323 H CDR1, V having the sequence of SEQ ID NO: 324 H CDR2 and V having the sequence of SEQ ID NO: 325 H CDR3; bb. V having the sequence of SEQ ID NO: 206 L CDR1, V having the sequence of SEQ ID NO: 207 L CDR2, V having the sequence of SEQ ID NO: 208 L CDR3, V having the sequence of SEQ ID NO: 326 H CDR1, V having the sequence of SEQ ID NO: 327 H CDR2 and V having the sequence of SEQ ID NO: 328 H CDR3; cc. V having the sequence of SEQ ID NO: 209 L CDR1, V having the sequence of SEQ ID NO: 210 L CDR2, V having the sequence of SEQ ID NO: 211 L CDR3, V having the sequence of SEQ ID NO: 329 H CDR1, V having the sequence of SEQ ID NO: 330 H CDR2 and V having the sequence of SEQ ID NO: 331 H CDR3; dd. V having the sequence of SEQ ID NO: 212 L CDR1, V having the sequence of SEQ ID NO: 213 L CDR2, V having the sequence of SEQ ID NO: 214 L CDR3, V having the sequence of SEQ ID NO: 332 H CDR1, V having the sequence of SEQ ID NO: 333 H CDR2 and V having the sequence of SEQ ID NO: 334 H CDR3; ee. V having the sequence of SEQ ID NO: 215 L CDR1, V having the sequence of SEQ ID NO: 216 L CDR2, V having the sequence of SEQ ID NO: 217 L CDR3, V having the sequence of SEQ ID NO: 335 H CDR1, V having the sequence of SEQ ID NO: 336 H CDR2 and V having the sequence of SEQ ID NO: 337 H CDR3; or.

9. The ABP according to any one of claims 6 to 8, comprising: a. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 76, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85; b. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 79, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 87; c. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 83; d. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 82; e. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 75, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84; f. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 86; g. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84; h. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 78, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 84; or i. a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

83.

10. The ABP of claim 1, which specifically binds to human CD69 and CD84.

11. The ABP of claim 10, wherein the ABP comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

36.

12. The ABP according to any one of claims 10 to 11, wherein the ABP comprises: V having the sequence of SEQ ID NO:45 L CDR1, V having the sequence of SEQ ID NO: 52 L CDR2, V having the sequence of SEQ ID NO: 55 L CDR3, V having the sequence of SEQ ID NO: 63 H CDR1, V having the sequence of SEQ ID NO: 66 H CDR2 and V having the sequence of SEQ ID NO: 71 H CDR3.

13. The ABP of claim 10, wherein the ABP comprises a light chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 74, and a heavy chain variable domain having at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, at least 97% or 100% sequence identity to the amino acid sequence of SEQ ID NO:

83.

14. The ABP of claim 1, wherein the ABP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-98.

15. The ABP of any one of claims 1 to 10, wherein the ABP comprises Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V domain antibody.

16. The ABP of claim 15, wherein the ABP is a Fab, Fab', F(ab')2, Fv, scFv, (scFv)2, a single chain antibody molecule, a dual variable domain antibody, a single variable domain antibody, a linear antibody, or a V domain antibody.

17. The ABP of any one of claims 1 to 16, wherein the ABP is a monoclonal antibody.

18. The ABP of any one of claims 1 to 17, wherein the ABP is selected from the group consisting of IgG, IgM, IgA, IgD and IgE antibodies.

19. The ABP of any one of claims 1 to 18, comprising a heavy chain constant region of IgG class and a subclass selected from IgGl, IgG2, IgG3 and IgG4.

20. The ABP of any one of claims 1 to 19, wherein the ABP is conjugated to a drug.

21. The ABP according to any one of claims 1 to 20, which is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC), wherein the ABP specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109, And wherein the ABP comprises a human Fc.

22. The ABP of claim 21, wherein the ABP is human, humanized or chimeric.

23. The ABP of claim 21 or 22, wherein the ABP is monoclonal.

24. The ABP of any one of claims 1 to 23, wherein the ABP is bispecific or multispecific.

25. The ABP of any one of claims 1 to 24, wherein the ABP comprises the heavy chain constant region of IgG.

26. The ABP of any one of claims 1 to 25, wherein the ABP is afucosylated.

27. The ABP of any one of claims 1 to 26, wherein: The ABP has a K of less than or equal to 50 nM, 10 nM, 5 nM, 1 nM, 0.5 nM or 0.1 nM as measured by a surface plasmon resonance (SPR) assay. D Binds to the target protein.

28. An ABP-drug conjugate comprising: the ABP according to any one of claims 1 to 27, a cytotoxic agent linked to the ABP, and optionally a linker linking the cytotoxic agent to the ABP.

29. The ABP-drug conjugate of claim 28, wherein the cytotoxic agent comprises an anti-angiogenic agent, a pro-apoptotic agent, an anti-mitotic agent, an anti-kinase agent, an alkylating agent, a hormone, a hormone agonist, a hormone antagonist, a chemokine, a drug, a prodrug, a toxin, an enzyme, an antimetabolite, an antibiotic, an alkaloid, or a radioisotope.

30. The ABP-drug conjugate of claim 28 or 29, wherein the linker is a cleavable linker or a non-cleavable linker.

31. A bispecific T cell engager (BiTE) comprising: an ABP according to any one of claims 1 to 27, and a T cell activation domain.

32. A pharmaceutical composition comprising the ABP of any one of claims 1 to 27, the ABP-drug conjugate of any one of claims 28-30, or the BiTE of claim 30, and an excipient.

33. An isolated polynucleotide or a group of isolated polynucleotides encoding the ABP of any one of claims 1 to 27 or the BiTE of claim 31.

34. A vector or a set of vectors comprising the isolated polynucleotide according to claim 33.

35. A host cell comprising the isolated polynucleotide according to claim 33 or the vector according to claim 34.

36. A method of producing an isolated antigen binding protein (ABP), comprising expressing the ABP in the host cell of claim 35 and isolating the ABP.

37. A chimeric antigen receptor (CAR), comprising: a. Extracellular antigen binding domain, b. transmembrane domain, c. signaling domain, and d. optionally at least one co-stimulatory domain, wherein the extracellular antigen binding domain specifically binds to a target protein selected from the group consisting of CD63, CD151, CD72, CD84, CD69 and CD109.

38. The CAR of claim 37, wherein the extracellular antigen binding domain comprises a single-chain variable fragment (scFv) of an antibody that specifically binds to the target protein.

39. The CAR of claim 37, wherein the extracellular antigen binding domain comprises the ABP of any one of claims 1 to 27.

40. The CAR of any one of claims 37 to 39, wherein the signaling domain comprises the intracellular domain of CD3ζ.

41. The CAR according to any one of claims 37 to 39, further comprising a costimulatory domain, wherein the costimulatory domain is a CD28 costimulatory domain, a 4-1BB costimulatory domain, a CD27 costimulatory domain, an OX40 costimulatory domain or an ICOS costimulatory domain.

42. The CAR of claim 41, wherein the co-stimulatory domain is a 4-1BB co-stimulatory domain.

43. The CAR of any one of claims 41 to 42, wherein the 4-1BB co-stimulatory domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:

105.

44. The CAR of any one of claims 37 to 39, wherein the transmembrane domain is a CD28 transmembrane domain.

45. The CAR of claim 44, wherein the CD28 transmembrane domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity with the amino acid sequence of SEQ ID NO:

100.

46. ​​The CAR of any one of claims 37 to 45, further comprising a hinge region.

47. The CAR of claim 46, wherein the hinge region is derived from a hinge region of a CD28 polypeptide.

48. The CAR of claim 47, wherein the hinge region comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:

99.

49. The CAR of any one of claims 37-48, wherein the signaling domain is a CD3 zeta signaling domain.

50. The CAR of claim 49, wherein the CD3 zeta signaling domain comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to the amino acid sequence of SEQ ID NO:

101.

51. The CAR of any one of claims 37 to 50, wherein the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 35-44.

52. The CAR of any one of claims 37 to 51, wherein the extracellular antigen binding domain specifically binds to human CD84.

53. A CAR according to any one of claims 37 to 51, wherein the extracellular antigen binding domain specifically binds to human CD69.

54. The CAR of any one of claims 37 to 53, wherein the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to an amino acid selected from SEQ ID NOs: 35-42.

55. The CAR of any one of claims 37-53, wherein the CAR comprises a polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acids of SEQ ID NO: 43, 44 or 36.

56. A polynucleotide encoding the CAR according to any one of claims 37-55.

57. The polynucleotide of claim 56, comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% sequence identity to the nucleotide sequence of SEQ ID NOs: 24-34.

58. A vector comprising the polynucleotide according to any one of claims 56 to 57.

59. An immune response cell expressing the CAR according to any one of claims 37 to 55.

60. An immune response cell comprising the polynucleotide according to claim 56 or the vector according to claim 58.

61. The immune response cell of any one of claims 59 to 60, wherein the immune response cell is an αβT cell, a γδT cell or a natural killer (NK) cell.

62. The immune response cell according to claim 61, wherein the αβ T cells are CD4 + T cells or CD8 + T cells.

63. A method for preparing an immune response cell according to any one of claims 59 to 62, the method comprising transfecting or transducing the polynucleotide according to claim 56 or the vector according to claim 58 into an immune cell.

64. The method of claim 63, wherein the method comprises expanding the immune cells for at least 48 hours.

65. The method of claim 64, wherein the immune cells are transduced at a multiplicity of infection of 1 to 100.

66. The method of claim 65, wherein the method further comprises, after transduction, washing the vector of claim 58, and expanding the transduced immune cells for at least 2 days.

67. A method of treating a subject, the method comprising: Administering a therapeutically effective amount of the ABP of any one of claims 1 to 27, the ABP-drug conjugate of any one of claims 28-30, the pharmaceutical composition of claim 32, or the immune response cell of any one of claims 59 to 62.

68. The method of claim 67, wherein the subject suffers from a myeloid disorder (MD) or acute leukemia (AL).

69. The method of claim 67, wherein the myeloid disorder (MD) and acute leukemia (AL) are of pediatric or adult onset.

70. The method of claims 67-69, wherein the ABP, the pharmaceutical composition, or the immune response cell is administered in combination with an additional agent.

71. The method of claim 70, wherein the additional agent is a chemotherapeutic agent or a biologic agent.

72. The method of claim 71, wherein the chemotherapeutic agent is selected from the group consisting of cytarabine, daunorubicin, idarubicin, cladribine, mitoxantrone, azacitidine, decitabine, and CPX-351 The group composed of.

73. The method of claim 70, wherein the additional agent is a Hedgehog signaling pathway inhibitor.

74. The method of claim 73, wherein the Hedgehog signaling pathway inhibitor is a Sonic Hedgehog signaling pathway inhibitor.

75. The method of claim 74, wherein the Sonic Hedgehog signaling pathway inhibitor is selected from the group consisting of vismodegib, sonidegi, and arsenic trioxide (ATO).

76. The method of claim 73, wherein the Hedgehog signaling pathway inhibitor is Daurismo TM ).

77. The method of claim 71, wherein the additional agent is a FMS-like tyrosine kinase 3 (FLT3) inhibitor.

78. The method of claim 77, wherein the FLT3 inhibitor is selected from midostaurin Gilteritinib The group consisting of sorafenib, lestaurinib, quizartinib, and clelanib.

79. The method of claim 73, wherein the additional agent is an isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) inhibitor.

80. The method of claim 79, wherein the IDH1 or IDH2 inhibitor is Ivosidenib Ensidipine 81. The method of claim 73, wherein the additional agent is a B-cell lymphoma 2 (BCL2) inhibitor.

82. The method of claim 81, wherein the BCL2 inhibitor is venetoclax 83. The method of claim 73, wherein the additional agent is a CD33 targeting agent.

84. The method of claim 83, wherein the CD33 targeting agent is gemtuzumab ozogamicin (Mylotarg TM ) or tariline-vadatuximab (SGN-CD33A).

85. The method of claim 73, wherein the additional agent is a cell cycle checkpoint inhibitor.

86. The method of claim 85, wherein the cell cycle checkpoint inhibitor is an Aurora kinase inhibitor, a Polo-like kinase 1 (PLK1) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, or a checkpoint kinase 1 (CHK1) inhibitor.

87. The method of claim 73, wherein the additional agent is an immune checkpoint inhibitor.

88. The method of claim 73, wherein the immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

89. The method of claims 67-69, further comprising the step of treating the subject with a chemotherapeutic agent or hematopoietic stem cells prior to administering the ABP, the pharmaceutical composition, or the immune response cells.

90. The method of claims 67 to 69, wherein the administering step is not followed by, or is not combined with, immunoglobulin therapy for hematopoietic rescue or autologous / allogeneic hematopoietic stem cell therapy (HSC).

91. The method of claim 90, wherein the immunoglobulin therapy is intravenous immunoglobulin (IVIG) therapy.

92. The method of any one of claims 67 to 91, wherein the subject has a refractory disease.

93. The method of any one of claims 67 to 92, wherein the subject has a recurrent disease.

94. The method of any one of claims 67 to 93, wherein the subject is an adult AML patient.

95. The method of any one of claims 67 to 93, wherein the subject is a pediatric AML patient.

96. The method of any one of claims 67 to 95, wherein the subject has been administered a chemotherapeutic agent or has undergone hematopoietic stem cell therapy prior to administering a therapeutically effective amount of the ABP, the pharmaceutical composition, or the immune response cells to the subject.

97. The method of any one of claims 67 to 95, wherein the subject is unresponsive to chemotherapy or hematopoietic stem cell therapy.

98. The method of any one of claims 67 to 97, wherein the subject has myeloblastic (M0) AML.

99. The method of any one of claims 67 to 97, wherein the subject has myeloblastic (M1) AML.

100. The method of any one of claims 67 to 97, wherein the subject has myeloblastic (M2) AML.

101. The method of any one of claims 67 to 97, wherein the subject has promyelocytic (M3) AML.

102. The method of any one of claims 67 to 97, wherein the subject has myelomonocytic (M4) AML.

103. The method of any one of claims 67 to 97, wherein the subject has monocytic (M5) AML.

104. The method of any one of claims 67 to 97, wherein the subject has erythroleukemia (M6) type AML.

105. The method of any one of claims 67 to 97, wherein the subject has megakaryocytic (M7) AML.

106. The method of any one of claims 67 to 105, wherein the immune response cells are administered to the subject at a dose of 100,000 cells / kg to 25 million cells / kg.

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