Terminal deoxynucleoside acyltransferase antibodies and uses thereof

Abstract

Aspects of the present disclosure relate to novel antibodies and antigen binding fragments. Further aspects relate to polypeptides comprising the antigen-binding fragments of the disclosure, and compositions comprising the polypeptides, antibodies, and / or antigen-binding fragments of the disclosure. Nucleic acids encoding the antibodies or antigen binding fragments of the disclosure are also described. In particular embodiments, the present disclosure provides immune compositions that target TdT peptides, including those associated with HLA-A2. Particular embodiments allow for the detection, diagnosis, treatment, and prevention of cancer in an individual in need thereof.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 336,912, filed on April 29, 2022, which is incorporated herein by reference in its entirety. [Background of the invention]

[0003] [I. Technical field]

[0004] Aspects of the present disclosure relate to at least the fields of immunology, cell biology, molecular biology, and medicine.

[0005] [II. Background technology]

[0006] Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in T-cell and B-cell progenitors that catalyzes the transfer of nucleotides during DNA rearrangement (Kunkel et al., 1986; Desiderio et al., 1984). TdT is expressed in up to 95% of T- and B-lineage ALL and is widely used as a diagnostic marker for these malignancies (Drexler et al., 1986). In addition, TdT expression is frequently detected in poorly differentiated AML (Patel et al., 2012; Drexler et al., 1993). Off-target activity of TdT in immature progenitors has been implicated in leukemogenesis (Borrow et al., 2019a; Borrow et al., 2019b). The absence of TdT expression in primitive hematopoietic progenitors or mature peripheral lymphocytes makes it an ideal therapeutic target for leukemia. [Summary of the Invention]

[0007] Various aspects of the present disclosure relate to novel antibodies and antigen-binding fragments, and methods of using these antibodies and functional fragments. Further aspects of the present disclosure relate to any type of polypeptide comprising an antigen-binding fragment, and compositions comprising the polypeptides, antibodies and / or antigen-binding fragments of the present disclosure. Nucleic acids encoding antibodies or antigen-binding fragments of the present disclosure are also described. The present disclosure also relates to nucleic acids encoding antibody heavy chains, wherein the nucleic acids have at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range thereof) sequence identity with one of SEQ ID NOs: 22 to 24. Also described are nucleic acids encoding light chains of antibodies of the disclosure, wherein the nucleic acids have at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity to one of SEQ ID NOs: 25 to 27. Further aspects relate to vectors or expression vectors comprising the nucleic acids of the disclosure and host cells comprising the polypeptides, nucleic acids, vectors, antibodies or antigen-binding fragments of the disclosure. The nucleic acids of the disclosure may be DNA or RNA.

[0008] Also described is a method of preparing a cell, the method comprising transferring one or more nucleic acids disclosed into a cell. In some embodiments, the method further comprises culturing the cell under conditions that allow expression of the polypeptide from the nucleic acid. In some embodiments, the method further comprises isolating the expressed polypeptide. The cell may be further defined as a human cell, an immune effector cell, a B cell, a T cell, a Chinese hamster ovary cell, a NS0 mouse myeloma cell, a PER.C6 cell, or any cell described herein.

[0009] A further aspect of the present disclosure relates to a method for treating or preventing cancer in a subject, the method comprising administering to the subject an antibody of the present disclosure or a composition comprising the antibody, an antigen binding fragment or a composition comprising the antigen binding fragment, a polypeptide, a nucleic acid or a host cell. However, a further aspect relates to a method for evaluating a sample from a subject, the method comprising contacting a biological sample from the subject or an extract thereof with at least one antibody, antigen binding fragment or polypeptide disclosed. A method for diagnosing cancer in a subject is also disclosed, the method comprising contacting a biological sample from the subject or an extract thereof with at least one antibody, antigen binding fragment or any polypeptide disclosed herein. In certain aspects, the disclosed composition is formulated as a vaccine for treating or preventing cancer. In some embodiments, the antibody, antigen binding fragment or composition of the present disclosure is used in a vaccine for preventing cancer in a subject who does not have cancer. In some embodiments, the antibody, antigen binding fragment or composition of the present disclosure is used to treat a subject with cancer.

[0010] A method for preparing a cell is also described, the method comprising transferring one or more nucleic acids disclosed into a cell. In some aspects, the method further comprises culturing the cell under conditions that allow expression of the polypeptide from the nucleic acid. In some aspects, the method further comprises separating the expressed polypeptide. A method for producing a polypeptide is described, comprising transferring one or more nucleic acids or vectors disclosed into a cell and separating the polypeptide expressed from the nucleic acid. The cell may be further defined as a human cell, an immune effector cell, a B cell, a T cell, a Chinese hamster ovary cell, a NS0 mouse myeloma cell, a PER.C6 cell, or any cell described herein.

[0011] A further aspect of the present disclosure relates to a method for treating or preventing cancer, the method comprising administering to a subject an antibody or a composition comprising the antibody, an antigen binding fragment or a composition comprising the antigen binding fragment, a polypeptide, a nucleic acid or a host cell of the present disclosure. However, a further aspect relates to a method for evaluating a sample from a subject, the method comprising contacting a biological sample or an extract thereof from a subject with at least one antibody or a composition comprising the antibody, an antigen binding fragment or a composition comprising the antigen binding fragment, or a polypeptide of the present disclosure. A method for diagnosing cancer in a subject is also disclosed, the method comprising contacting a biological sample or an extract thereof from a subject with at least one antibody or a composition comprising the antibody, an antigen binding fragment or a composition comprising the antigen binding fragment, or any polypeptide of the present disclosure. In certain aspects, the disclosed composition is formulated as a vaccine for treating or preventing cancer. In certain aspects, an antibody or a composition comprising the antibody, an antigen binding fragment or a composition comprising the antigen binding fragment, or a disclosed composition is used in a vaccine for preventing cancer in a subject without cancer. In certain aspects, the antibodies or compositions comprising the antibodies, antigen-binding fragments or compositions comprising the antigen-binding fragments or compositions of the present disclosure are used to treat a subject having cancer.

[0012] Certain aspects of the present disclosure relate to antibodies or antigen-binding fragments comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a specific sequence, wherein the light chain variable region comprises a specific sequence. Further aspects relate to antibodies or antigen-binding fragments comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises at least 80% sequence identity to SEQ ID NOs: 22-24 or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity. Certain aspects relate to antibodies or antigen-binding fragments comprising a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises at least 80% sequence identity to SEQ ID NOs: 25-27 or has at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein).

[0013] Various aspects of the present disclosure relate to antibodies or antigen-binding fragments comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of a heavy chain variable region from an antibody clone of B1, G7 or G9, and wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of a light chain variable region from the same corresponding clone of B1, G7 or G9. A further aspect relates to an antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2 and HCDR3 having at least 80% sequence identity to, or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to, a heavy chain variable region from a B1, G7 or G9 antibody clone (or wherein 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the light chain variable region of the same corresponding clone of B1, G7 or G9.

[0014] Various aspects of the present disclosure relate to antibodies, antigen-binding fragments or polypeptides comprising a heavy chain variable region having HCDR1, HCDR2 and HCDR3, and a light chain variable region having LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 40 to 42, and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 43, 45 or "DDN".

[0015] The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 can be determined from the variable region sequence by methods known in the art. In some aspects, the CDR is the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 determined by the Chothia method. In some aspects, the CDR is the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 determined by the Kabat method. In some aspects, the CDR is the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 determined by the IMGT method.

[0016] The polypeptide of the present disclosure may comprise at least two antigen binding fragments, wherein each antigen binding fragment is independently selected from the antigen binding fragments of the present disclosure. In some aspects, the polypeptide is multivalent. In some aspects, the polypeptide is multispecific. In some aspects, the polypeptide is bispecific. In some aspects, the polypeptide comprises, at least comprises, or comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigen binding regions. Each antigen binding region can be independently selected from the antigen binding regions of the present disclosure. In some aspects, the polypeptide may have repeating units of the same antigen binding region, such as at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeating units.

[0017] In certain aspects, the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with the heavy chain variable region of antibody clone B1, G7 or G9 and / or the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with the light chain variable region of antibody clone B1, G7 or G9. In certain aspects, the heavy chain variable region comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity with the heavy chain variable region of antibody clone B1. and / or the light chain variable region comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity with the light chain variable region of the same antibody clone B1. In certain aspects, the heavy chain variable region comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the heavy chain variable region of antibody clone G7. and / or the light chain variable region comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity with the light chain variable region of the same antibody clone G7.In certain aspects, the heavy chain variable region comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the heavy chain variable region of antibody clone G9. and / or the light chain variable region comprises an amino acid sequence having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity with the light chain variable region of the same antibody clone G9.

[0018] In certain aspects, the heavy chain variable region comprises a heavy chain framework region having antibody clone B1 or a heavy chain framework region having at least 80% sequence identity thereto, and the light chain variable region comprises a light chain framework region having the same antibody clone B1 or a light chain framework region having at least 80% sequence identity thereto. In certain aspects, the heavy chain variable region comprises a heavy chain framework region having antibody clone B1 or a heavy chain framework region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity thereto. , and the light chain variable region comprises a light chain framework region having the same antibody clone B1 or a light chain framework region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity thereto.

[0019] In certain aspects, the heavy chain variable region comprises a heavy chain framework region having antibody clone G7 or a heavy chain framework region having at least 80% sequence identity thereto, and the light chain variable region comprises a light chain framework region having the same antibody clone G7 or a light chain framework region having at least 80% sequence identity thereto. In certain aspects, the heavy chain variable region comprises a heavy chain framework region having antibody clone G7 or a heavy chain framework region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity thereto. , and the light chain variable region comprises a light chain framework region having the same antibody clone G7 or a light chain framework region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity thereto.

[0020] In certain aspects, the heavy chain variable region comprises a heavy chain framework region having antibody clone G9 or a heavy chain framework region having at least 80% sequence identity thereto, and the light chain variable region comprises a light chain framework region having the same antibody clone G9 or a light chain framework region having at least 80% sequence identity thereto. In certain aspects, the heavy chain variable region comprises a heavy chain framework region having antibody clone G9 or a heavy chain framework region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity thereto. , and the light chain variable region comprises a light chain framework region having the same antibody clone G9 or a light chain framework region having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% (or any derivable range therein) sequence identity thereto.

[0021] The disclosed antibodies or antigen-binding fragments can be human, chimeric or humanized. In certain aspects, the antibodies or antigen-binding fragments bind to the TdT peptide with a kD of about 10 -3 M~about 10 -9 In certain aspects, the antibody or antigen-binding fragment binds to the TdT peptide with a kD of about, at least, or at most 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 , 10 -13 , 10 -14 , 10 -15 , 10 -16 , 10 -17 or 10 -18 (or any derivable range therein) M. In some aspects, the antibody or antigen binding fragment specifically binds to the TdT peptide. The antibody can be further defined as a neutralizing antibody. In some aspects, the antibody or antigen binding fragment is further defined as a human antibody or antigen binding fragment, a humanized antibody or antigen binding fragment, a recombinant antibody or antigen binding fragment, a chimeric antibody or antigen binding fragment, an antibody or antigen binding fragment derivative, a modified antibody or antigen binding fragment, a bispecific antibody, a monoclonal antibody or antigen binding fragment, a single domain antibody or a single chain antibody. In some aspects, the antigen binding fragment is further defined as a single chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv or rIgG. In some aspects, the antibody, antigen binding fragment or polypeptide is operably connected to a detectable marker. Detectable labels are included herein.

[0022] Various aspects of the present disclosure also relate to multispecific and / or multivalent antibodies and polypeptides. Thus, aspects relate to bivalent or bispecific antibodies comprising two antigen binding fragments, wherein the antigen binding fragments are two identical antigen binding fragments or two different antigen binding fragments as described herein. The present disclosure also provides for multispecific polypeptides. Aspects relate to polypeptides comprising or comprising at least 2, 3, 4, 5 or 6 antigen binding fragments.

[0023] The antigen binding fragment may be at least 2, 3, 4, 5 or 6 scFv, F(ab')2, Fab', Fab, Fv or rIgG, or a combination thereof. The polypeptide and / or antigen binding fragment of the present disclosure may include a linker between the heavy chain and light chain variable regions or between the antigen binding fragments. The linker may be a flexible linker. Typical flexible linkers include glycine polymers (G)n or glycine-serine polymers. Glycine-alanine polymers, alanine-serine polymers and other flexible linkers known in the art and can be used as linkers in the polypeptides of the present disclosure. Specific examples include (GGGS)n (SEQ ID NO: 46) or (GGGGS)n (SEQ ID NO: 47), wherein n = 1 to 10, and further specific examples include GGGSGGGS (SEQ ID NO: 48) or GGGSGGGGSG GGGS (SEQ ID NO: 49).

[0024] Compositions of the present disclosure, such as pharmaceutical compositions, may include pharmaceutical excipients, carriers or molecules described herein. In certain aspects, the composition further comprises an adjuvant or immunostimulant. Such adjuvants or immunostimulants may include, but are not limited to, pattern recognition receptors, such as Toll-like receptors, RIG-1 and NOD-like receptors (NLRs), mineral salts such as alum, alum bound to monophosphoryl lipid (MPL) A of Enterobacteria (e.g., Escherichia coli, Salmonella minnesota, Salmonella typhimurium or Shigella flexneri), or alum specifically bound to MPL (ASO4) and MPL A of the above-mentioned bacteria, respectively, saponins such as QS-21, Quil-A, ISCOMs, ISCOMATRIX, emulsions such as MF59, Montanide, ISA 51 and ISA 720, AS02 (QS21 + squalene + MPL), liposomes and liposome preparations, such as AS01, synthetic or specially prepared microparticles and microcarriers, such as outer membrane vesicles (OMV) of bacterial origin of Neisseria gonorrhoeae, Chlamydia trachomatis, etc., or chitosan particles, storage adjuvants, such as Pluronic block copolymers, specifically modified or prepared peptides, such as muramyl dipeptide, aminoalkyl glucosamine 4-phosphate, such as RC529, or proteins, such as bacterial toxins or toxin fragments). The composition may contain more than one antibody and / or antigen-binding fragment disclosed. Therefore, the composition of the present disclosure may include, at least include, or at most include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 antibodies and / or antigen-binding fragments disclosed. The composition of the present disclosure can be formulated for the administration routes described herein. In certain aspects, the composition, antibody, antigen-binding fragment or polypeptide is formulated for parenteral, intravenous, subcutaneous, intramuscular or intranasal administration. In a specific aspect, the composition is formulated for intranasal administration.

[0025] In some aspects, the host cell is a human cell, an immune effector cell, an immune cell, a B cell, a T cell, a Chinese hamster ovary cell, a NS0 mouse myeloma cell or a PER.C6 cell. In some aspects, the host cell is a cell type or a cell population described herein.

[0026] In aspects of the present disclosure, the subject or patient can be a human subject or a human patient. In some aspects, the subject or patient is a non-human animal. In some aspects, the non-human animal is a bat, monkey, camel, rat, mouse, rabbit, goat, chicken, bird, cat, horse or dog. Compared with the general population, the subject can be further defined as a high-risk subject. High-risk subjects include those who may be smokers, have a personal or family history of cancer, have been exposed to carcinogens, are older than 50, 55, 60, 65 or 70 years old, etc. In some aspects, the subject has one or more cancer symptoms. Examples of cancer symptoms include fatigue or extreme tiredness that does not improve with rest; unexplained weight loss or gain of more than 10 pounds; eating problems, such as not feeling hungry, difficulty swallowing, abdominal pain, or nausea and vomiting; swelling or lumps anywhere on the body; thickening or lumps in the breasts or other parts of the body; pain, especially new or unexplained pain, that does go away or gets worse; skin changes, such as lumps that bleed or become scaly, new moles or changes in moles, sores that don't heal, or yellowing of the skin or eyes (jaundice); cough or hoarseness that doesn't go away; unusual bleeding or bruising that doesn't go away; changes in bowel habits, such as constipation or diarrhea that doesn't go away, or changes in the appearance of stool; bladder changes, such as pain when urinating, blood in the urine, or the need to urinate more or less; fever or night sweats; headache; and / or vision or hearing problems. In some aspects, the subject does not have any symptoms of cancer. In some aspects, the subject has been diagnosed with cancer. In some aspects, the subject has not been diagnosed with cancer. In some aspects, the subject has been previously treated for cancer. In some aspects, the subject has not been previously vaccinated with a cancer vaccine. In some aspects, the prior treatment includes surgery, radiation therapy, chemotherapy, drug therapy, hormone therapy, or a combination thereof.

[0027] In certain aspects of the present disclosure, the method further comprises incubating the antibody, antigen binding fragment or polypeptide under conditions that allow the antibody, antigen binding fragment or polypeptide to bind to an antigen in a biological sample or an extract thereof. In certain aspects, the method further comprises detecting the binding of an antigen to the antibody, antigen binding fragment or polypeptide. In certain aspects, the method further comprises contacting the biological sample with at least one capture antibody, antigen or polypeptide. The at least one capture antibody, antigen binding fragment or polypeptide may be an antibody, polypeptide or antigen binding fragment of the present disclosure. In certain aspects, the capture antibody is connected to a solid support or is operably connected to a solid support. The term "operably connected" refers to a situation where two components are combined or can be combined to form a complex. For example, the components may be covalently linked and / or located on the same polypeptide, such as in a fusion protein, or there may be a certain degree of binding affinity between the components, such as binding affinity occurring through van der Waals forces. In certain aspects, the biological sample comprises a blood sample, a urine sample, a stool sample, a nasopharyngeal sample, a cerebrospinal fluid sample, a cheek scrape sample, a nipple aspiration sample, a biopsy sample, or a combination thereof. In aspects of the present disclosure, at least one antibody, Fab or polypeptide is operably linked to a detectable label. In some aspects, the method further comprises incubating the antibody, Fab or polypeptide under conditions that allow the antibody, Fab or polypeptide to bind to an antigen in a biological sample or an extract thereof. In some aspects, the method further comprises detecting the binding of an antigen to the antibody, Fab or polypeptide. In some aspects, the method further comprises contacting the biological sample with at least one capture antibody, antigen or polypeptide.

[0028] Various aspects of the disclosure relate to antibodies, antigen-binding fragments, or polypeptides comprising the heavy chain variable region and the light chain variable region of SEQ ID NOs: 22 and 25, respectively. Various aspects of the disclosure relate to antibodies, antigen-binding fragments, or polypeptides comprising the heavy chain variable region and the light chain variable region of SEQ ID NOs: 23 and 26, respectively. Various aspects of the disclosure relate to antibodies, antigen-binding fragments, or polypeptides comprising the heavy chain variable region and the light chain variable region of SEQ ID NOs: 24 and 27, respectively.

[0029] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. In addition, the compositions of the invention can be used to implement the methods of the invention.

[0030] Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

Brief Description of the Figures

[0032] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments given herein.

[0033] Figure 1 : Expression of TdT in normal and malignant lymphocytes.

[0034] Figure 2 : Targeting TdT with chimeric TCR and BiTE.

[0035] Figure 3 A-3E: Structure, expression and function of TdT-specific CAR and cTCR. Figure 3 A) Schematic diagram showing the structure of CAR and cTCR. Figure 3 B) Expression of TdT.CAR and cTCR in primary T cells (n=7 donors). Figure 3 C) Expansion of modified T cells after transduction (n=7). Figure 3 D) Expression of CD27 and CD45RA on control and TdT-specific CD8+ T cells (n=6). Figure 3 E) Cytotoxicity of TdT-specific T cells against BV173 cells, co-cultured at an E:T ratio of 1:4 for 3 days (n=3).

[0036] Figure 4 A~4B: Specificity of TdT.cTCR T cells. ( Figure 4 A) Cytotoxicity of cTCR T cells against BV173 (TdT+ / A2+), NALM6 (TdT+ / A2+), CCRF-CEM (TdT+ / A2-) and THP-1 (TdT- / A2+) cells after 3 days of co-culture (n=3-4). Figure 4 B) Viability of normal autologous T and B cells after co-culture with TdT.cTCR- or CD19.CAR-expressing T cells was measured by Annexin V / 7-AAD staining (n=3).

[0037] Figure 5 A-5B: Effector function of CD4+ and CD8+ T cells transduced with TdT.cTCR. Representative flow graphs showing degranulation after co-culture with parental and MHC I-deficient (b2MKO) BV173 leukemia cells ( Figure 5 A) and IFNγ / TNFα production ( Figure 5 B).

[0038] Figure 6: Redirection of T cell cytotoxicity against TdT+ / A2+ cell lines by CAR, cTCR or BiTE (n=3).

[0039] Figure 7 One embodiment of a chimeric TCR is shown, wherein the V H and V L The domains are present on a single TCRα chain, in contrast to other receptors in the art (Liu et al., Sci Transl Med. 2021; 13(586):eabb5191). The constant chains of mouse TCRα and TCRβ are described.

[0040] Figure 8 Represents the expression of cTCR and STAR on T cells after retroviral transduction. NT: not transduced.

[0041] Fig. 9 The cytotoxicity of cTCR- and STAR-expressing T cells against the BV173 cell line (TdT+, HLA-A2+) was demonstrated.

[0042] Fig.10 Shown is the expression of cTCR and BiTE on T cells after retroviral transduction. NT: not transduced.

[0043] Fig.11 We demonstrated cTCR T cell cytotoxicity and BiTE-mediated killing of the BV173 cell line (TdT+, HLA-A2+). [Detailed description of the invention]

[0045] [I. Definition example]

[0046] In this application, the term "about" is used to indicate a value that includes the inherent variation of error for the measurement or quantification method employed.

[0047] When used with the term "comprising," the word "a" or "an" can mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0048] The phrase "and / or" means either "and" or "or." To clarify, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and / or" operates as an inclusive or.

[0049] As used herein, the phrase "at least one of" when used with a list of items means that different combinations of one or more of the listed items may be used, and only one of the items in the list may be required. An item may be a specific object, thing, step, operation, process, or category. In other words, "at least one of" means that any combination of items or numbers of items in the list may be used, but not all items in the list are required. For example, but not limited to, "at least one of item A, item B, or item C" means item A; item A and item B; item B; item A, item B, and item C; item B and item C; or items A and C. In some cases, "at least one of items A, B, or C" means, but is not limited to, two A items, one B item, and ten C items; 4 B items and 7 C items; or some other suitable combination.

[0050] In this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to mean the inclusion of the stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase and is limited to only those other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but no other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0051] In this specification, references to "one embodiment", "an embodiment", "a specific embodiment", "a related embodiment", "an embodiment", "an additional embodiment" or "a further embodiment" or a combination thereof, mean that a particular feature, structure or characteristic associated with the embodiment is included in at least one embodiment of the present invention. Therefore, the appearance of the above phrases in different places in this specification does not necessarily refer to the same embodiment. In addition, specific features, structures or characteristics may be combined in any appropriate manner in various embodiments.

[0052] The compositions and methods used therein may "comprise," "consist essentially of, or consist of" any ingredient or step disclosed throughout the specification. Compositions and methods consisting "essentially of" any disclosed ingredient or step limit the scope of the claim to specific materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. In this specification and claims, "comprising" (and any forms of inclusion, such as "comprise" and "comprises"), "having" (and any forms of having, such as "have" and "has"), "including" (and any forms of inclusion, such as "includes" and "include"), or "containing" (and any forms of inclusion, such as "contains" and "contain") are inclusive or open-ended and do not exclude other, uncited elements or method steps. It is contemplated that embodiments described herein in the context of the term "comprising" may also be implemented in the context of the term "consisting of" or "consisting essentially of."

[0053] The term "peptide" as used herein generally refers to amino acids connected by peptide bonds. A peptide may comprise an amino acid chain of about 5 to 50 residues. A peptide may comprise an amino acid chain shorter than 10 residues, including oligopeptides, dipeptides, tripeptides, and tetrapeptides. A peptide may comprise a chain longer than 50 residues and may be referred to as a "polypeptide" or "protein". In a specific embodiment, the term refers to the TdT peptide of ALYDKTKRI (SEQ ID NO: 3) or any peptide having at least 70, 75, 80, 85, 86, 87, 88, 89, 90 or more% identity with SEQ ID NO: 3. The peptide may be at least 5, 6, 7, 8 or all consecutive residues of SEQ ID NO: 3. There may be one or two substitutions compared to SEQ ID NO: 3. In a specific embodiment, any antibody contained herein is capable of targeting these variant peptides.

[0054] The term "subject" as used herein generally refers to an animal, such as a mammal (e.g., a human). For example, a subject may include a vertebrate, a mammal, a rodent (e.g., a mouse), a primate, a monkey, or a human. Animals may include, but are not limited to, farm animals, sports animals, and pets, including cats, dogs, and horses. A subject may include a healthy or asymptomatic individual, an individual suffering from or suspected of having a disease (e.g., cancer) or a susceptibility to the disease, and / or an individual in need of treatment or suspected of needing treatment. A subject may be a patient. "Individual," "subject," and "patient" may be used interchangeably and may refer to a person or a non-person.

[0055] "Treatment" or treatment of a disease or condition refers to the implementation of a regimen, which may include the administration of one or more drugs to an individual (e.g., a patient) in an effort to alleviate the signs or symptoms of the disease. Desirable therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. Relief may occur before signs or symptoms of the disease or condition appear, or after the disease or condition appears. Thus, "treating" or "treatment" may include "prophylaxis" or "prevention" of a disease or adverse condition. In addition, "treating" or "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes regimens that have only a marginal effect on the patient.

[0056] The term "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the health of a subject with respect to medical treatment of the condition. This includes, but is not limited to, a reduction in the frequency or severity of one or more signs or symptoms of a disease, including cancer, and / or a delay in its onset or spread.

[0057] It is specifically contemplated that any limitation discussed about one embodiment of the present invention may be applicable to any other embodiment of the present invention. In addition, any composition of the present invention may be used for any method of the present invention, and any method of the present invention may be used to produce or utilize any composition of the present invention. The various aspects of the embodiments set forth in the examples are also embodiments that can be implemented in the context of embodiments discussed elsewhere in the application (e.g., in the description of brief overview, detailed description, claims, and brief description of the accompanying drawings) and elsewhere in different embodiments.

[0058] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described using "use" claim language, such as "use" of any compound, composition, or agent discussed herein to achieve or carry out the described therapeutic, diagnostic, or physiological purpose or effect.

[0059]

II. TdT peptide-specific antibody

[0060] Various aspects of the present disclosure relate to antibodies, antigen-binding fragments thereof, or polypeptides that are capable of specifically binding to a TdT peptide. Certain aspects relate to antibodies or fragments thereof that specifically bind to a TdT peptide. In specific embodiments, the antibody or functional fragment thereof binds to a TdT peptide that comprises, consists of, or consists essentially of SEQ ID NO: 3 or a variant thereof that has at least 80, 85, 88, 90, or more % identity to the peptide.

[0061] The term "antibody" refers to an intact immunoglobulin of any isotype or a fragment thereof that is capable of competing with the intact antibody for specific binding to a target antigen, and includes chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably to refer to any of several classes of structurally related proteins that function as part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM, and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen binding activity.

[0062] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent, such as an antibody. An antigen may have one or more epitopes that can interact with different antibodies.

[0063] The term "epitope" includes any region or portion of a molecule that is capable of inducing an immune response by binding to an immunoglobulin or T-cell receptor. Epitope determinants may include chemically active surface groups such as amino acids, sugar side chains, phosphate groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. Typically, an antibody specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture.

[0064] Many different epitope mapping techniques recognized in the art can be used to identify the epitope region of a given polypeptide, including: X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, for example, epitope mapping schemes (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY Such techniques are known in the art, for example, in U.S. Patent No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82: 178-182 (1985); Geysen et al. Molec. Immunol. 23: 709-715 (1986) described. In addition, standard antigenicity and hydrophilicity maps can also be used to predict and identify the antigenic region of a protein.

[0065] The term "immunogenic sequence" refers to a molecule comprising an amino acid sequence of at least one epitope, such that the molecule is capable of stimulating the production of antibodies in an appropriate host. The term "immunogenic composition" refers to a composition comprising at least one immunogenic molecule (eg, an antigen or carbohydrate).

[0066] A complete antibody is usually composed of two full-length heavy chains and two full-length light chains, but may include fewer chains in some cases, such as antibodies naturally present in camelids that may only contain heavy chains. The antibodies disclosed herein may come from only a single source, or may be "chimeras", i.e., different parts of the antibodies may come from two different antibodies. For example, the variable region or CDR region may come from rat or mouse sources, while the constant region comes from different animal sources, such as humans. Antibodies or binding fragments can be produced in hybridomas by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. Unless otherwise indicated, the term "antibody" includes derivatives, variants, fragments, and mutant proteins thereof, examples of which are described below (Sela-Culang et al., Front Immunol. 2013; 4: 302; 2013).

[0067] The term "light chain" includes full-length light chains and fragments thereof, wherein the fragments have sufficient variable region sequence to confer binding specificity. The molecular weight of a full-length light chain is approximately 25,000 daltons and includes a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, designated as κ and λ. The term "VL fragment" refers to a fragment of a monoclonal antibody light chain, including all or part of the light chain variable region, including CDRs. The VL fragment may also include a light chain constant region sequence. The variable region domain of the light chain is located at the amino terminus of the polypeptide.

[0068] The term "heavy chain" includes full-length heavy chains and fragments thereof, which have sufficient variable region sequences to confer binding specificity. The molecular weight of a full-length heavy chain is approximately 50,000 Daltons, and includes one variable region domain (abbreviated herein as VH) and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" refers to a heavy chain fragment of a monoclonal antibody, including all or part of the heavy chain variable region, including CDRs. The VH fragment may also include a heavy chain constant region sequence. The number of heavy chain constant region domains will depend on the isotype. The VH domain is located at the amino terminus of the polypeptide, and the CH domain is located at the carboxyl terminus, with CH3 being closest to the -COOH terminus. The isotype of an antibody may be IgM, IgD, IgG, IgA, or IgE, defined by the presence of heavy chains, of which there are five categories: μ, δ, γ, α, or ε chains. There are several subtypes of IgG, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0069] In specific embodiments, the disclosure includes antibodies that bind a portion of a TdT protein, including antibodies that bind a TdT peptide comprising, consisting of, or consisting essentially of SEQ ID NO: 3. Although the antibody can be of any class, in specific embodiments, the antibody comprises a single chain variable fragment (scFv).

[0070] In various embodiments, the disclosure provides a plurality of scFvs, which can be used alone or in any combination. In a specific embodiment, the antibody is used for any type of chimeric polypeptide, including any receptor and / or any bispecific or multispecific antibody, including any bispecific T cell engager (BiTE). In a specific embodiment, the antibody comprises the following representative sequence:

[0071]

Clone 1: TdT_B1 polynucleotide sequence

[0072] ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG

[0073] CCGGCTCAAG TTCAGCTGGT GCAGAGCGGA GCGGAGGTGA AGAAGCCAGG GTCTTCCGTT

[0074] AAGGTCTCTT GTAAGGCCTC AGGCGGAACT TTCTCATCTT ACGCTATTTC CTGGGTAAGG

[0075] CAAGCACCAG GACAGGGATT AGAATGGATG GGGGGGATTA TCCCCATTTT CGGTACCGCG

[0076] AACTACGCCC AAAAATTTCA AGGTCGGGTA ACCATCACCG CCGATGAATC GACATCTACC

[0077] GCCTACATGG AACTGAGCTC TCTTCGGAGC GAGGACACCG CAGTCTATTA CTGCGCGAGA

[0078] GACGGGTATA GTGGTAGCTA CTACTATTAT TACGGCATGG ACGTTTGGGG CCAGGGGACC

[0079] TTAGTGACGG TGAGCAGCGG AGGAGGCGGC GGGTCCCAGT CTGCTCTGAC CCAACCTGCC

[0080] AGCGTCTCCG GCAGTCCAGG ACAAAGTATC ACCATCAGCT GCACTGGGAC TTCCAGCGAT

[0081] GTGGGGGGCT ACAATTACGT AAGTTGGTAC CAGCAACACC CTGGGAAGGC CCCCAAGCTG

[0082] ATGATTTATG ATGTCTCATA CAGACCGAGC GGAGTGAGCC ACCGTTTTAG TGGCTCCAAG

[0083] TCCGGAAACA CCGCTTCACT AACAATCAGT GGCTTACAAG CAGAGGACGA AGCCGATTAT

[0084] TACTGCTCAT CTTACACCAG TTCCAGTACG CTTGTGTTTG GTACTGGAACAAAGTTGACTGTGCTGGGC (SEQ ID NO: 4)

[0085]

Clone 1: TdT_B1 polypeptide sequence; signal peptide-V H -V L

[0086]

[0087] Among them, the signal peptide is located at the N-terminus and lacks a tag, V H The domain is underlined, and V L Domains are both italicized and underlined.

[0088] In the specific embodiment of B1, the V H and V L as follows:

[0089]

V H

[0090] AQVQLVQSGA EVKKPGSSVK VSCKASGGTF SSYAISWVRQ APGQGLEWMG GIIPIFGTAN

[0091] YAQKFQGRVT ITADESTSTA YMELSSLRSE DTAVYYCARD GYSGSYYYYY GMDVWGQGTLVTVSS(SEQ ID NO: 22)

[0092]

V L

[0093] QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMIYDVSYRPSGVSHRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTLV FGTGTKLTVL (SEQ ID NO: 25)

[0094] In a specific embodiment, the complementarity determining regions (CDRs) of B1 are as follows:

[0095] HCDR1: GGTFSSYA (SEQ ID NO: 28)

[0096] HCDR2: IIPIFGTA (SEQ ID NO: 29)

[0097] HCDR3: ARDGYSGSYY YYYGMDV (SEQ ID NO: 30)

[0098] LCDR1: SSDVGGYNY (SEQ ID NO: 31)

[0099] LCDR2: DVSLCDR3: SSYTSSSTLV (SEQ ID NO: 33)

[0100]

Clone 2: TdT_G7 polynucleotide sequence

[0101] ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG

[0102] CCGGCCCAAG TCCAGCTGGT TGAAAGCGGG GGAGGAGTGG TTCAGCCCGG AAGGAGTTTG

[0103] CGGTTGAGTT GTGCAGCATC AGGATTCACA TTTTCTTCCT ATGCAATGTC CTGGGTGCGT

[0104] CAGGCTCCCG GAAAGGGACT GGAATGGGTT AGCGCTATAT CTGGCAGCGG AGACTCTACA

[0105] TATTATGCTG ATTCAGTCAA AGGCCGTTTC ACCATCAGCA GAGACAACAG TAAGAATACC

[0106] CTCTACCTTC AGATGAACTC GCTACGTGCT GAAGACACCG CCGTTTACTA CTGTGCTAAA

[0107] GATCTCGACT CCTCAAGTCC GGACGATGCT TTTGACATCT GGGGACAAGG CACCACTGTC

[0108] ACCGTCAGCA GCGGAGGCGC CGCGCTGGCC GAAGTGGCAG CCGCGGTGGC TGACCCTATC

[0109] GTTCTGACTC AGTCTCCTGG AAAGCTGAGC CTCTCCCCAG GGGAGCGGGC CACTTTGTCC

[0110] TGTGGCGCGT CGCAGAGCGT ATCATCCAAT TACCTGGCAT GGTACCAGCA GAAACCTGGC

[0111] CTGGCTCCTC GGCTTTTGAT TTATGATGCT AGTATTAGGG CTACTGGCGT GCCTGATCGG

[0112] TTCTCGGGAT CAGGGAGCGC GACTGACTTC ACTTTGACGA TCAGCCGGCT GGACCCTGAG

[0113] GATTTTGCGG TGTACTATTG CCACCAGTAT TCATCTGCCC CCATGACTTTTGGACAGGGCACTAAGCTGG AGATTAAGAG G(SEQ ID NO:6)

[0114]

Clone 2: TdT_G7 Polypeptide Sequence

[0115]

[0116]

[0117] The signal peptide is located at the N-terminus and lacks a tag. H The domain is underlined, and V L Domains are both italicized and underlined.

[0118] In a specific embodiment of G7, V H and V L as follows:

[0119]

V H

[0120]

[0121]

V L

[0122]

[0123] In a specific embodiment, the complementarity determining regions (CDRs) of G7 are as follows:

[0124] HCDR1: GTFFSSYA (SEQ ID NO: 34)

[0125] HCDR2: ISGSGDST (SEQ ID NO: 35)

[0126] HCDR3: AKDLDSSSPD DAFDI (SEQ ID NO: 36)

[0127] LCDR1: QSVSSNY (SEQ ID NO: 37)

[0128] LCDR2: DASLCDR3: HQYSSAPMT (SEQ ID NO: 39)

[0129]

Clone 3: TdT_G9 polynucleotide sequence

[0130] ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG

[0131] CCGGCTCAAG TGCAGCTGGT CGAATCTGGA GCTGGGACTA AGAAGCCTGG CGAGAGTCTG

[0132] AAGATCTCTT GTAAGGCCAG CGGCTACAAC TTTGCTTCTT ACTGGATCGG GTGGGTTAGA

[0133] CAGATGCCTG GAAAAGGACT TGAGTGGGTA GGGATCATTG ATCCTAGCGA TTCCGATACC

[0134] AAATACAGCC CAAGCTTTGA AGGGCAAGTC ACTATTAGTG TGGACAAGTC TATCAGCACT

[0135] GCTCACCTGC AGTGGAGTAG CCTAGATGCG AGCGACACGG CCATGTACTA CTGCGCGAGG

[0136] AGTCTGGGCA GCTACTATGG AGATTGGTAT TTCGATCTCT GGGGGAGGGG CACACTGGTG

[0137] ACCGTGTCAT CCGGAGGAGG CGGCGGGTCC AACTTTATGC TGACCCAGCC ACATTCAATG

[0138] TCTGAAAGCC CTGGCAAAAC CGTAACAATC TCGTGCACAC GGTCAAGCGG GAGCATCGCC

[0139] AACAACTATG TTCAGTGGTA TCAGCAGAGA CCCGGGAGTT CCCCTACCAC TGTCATCTAC

[0140] GATGACAATC AGAGACCATC CGGGGTGCCC GACCGCTTCA GCGGCTCAAT AGATAGCTCC

[0141] AGCAACTCCG CCTCTTTAAC AATCTCGGGC CTGAAAACCG AGGACGAAGC TGATTATTAC

[0142] TGCCAGTCCT ATGACTCCAG CAACGTGATT TTTGGTGGAG GGACTAAACT GACCGTCCTC GGA(SEQ ID NO:8)

[0143]

Clone 3: TdT_G9 Polypeptide Sequence

[0144]

[0145] Among them, the signal peptide is located at the N-terminus and lacks a tag, V H The domain is underlined, and V L Domains are both italicized and underlined.

[0146] In a specific embodiment of G9, the V H and V L as follows:

[0147]

V H

[0148]

[0149]

V L

[0150]

[0151] In a specific embodiment, the complementarity determining regions (CDRs) of G9 are as follows:

[0152] HCDR1: GYNFASYW (SEQ ID NO: 40)

[0153] HCDR2: IDPSDSDT (SEQ ID NO: 41)

[0154] HCDR3: ARSLGSYYGD WYFDL (SEQ ID NO: 42)

[0155] LCDR1:SGSIANNY(SEQ ID NO:43)

[0156] LCDR2: DDN

[0157] LCDR3: QSYDSSNVI (SEQ ID NO: 45)

[0158] Thus, in a specific embodiment, any antibody used herein may comprise the following V H One of the sequences or one or more modifications thereof:

[0159] (1) AQVQLVQSGA EVKKPGSSVK VSCKASGGTF SSYAISWVRQ APGQGLEWMGGIIPIFGTANYAQKFQGRVT ITADESTSTA YMELSSLRSE DTAVYYCARD GYSGSYYYYYGMDVWGQGTL VTVSS (SEQ IDNO: 22);

[0160] or

[0161] (3) AQVQLVESGA GTKKPGESLK ISCKASGYNF ASYWIGWVRQ MPGKGLEWVGIIDPSDSDTKYSPSFEGQVT ISVDKSISTA HLQWSSLDASDTAMYYCARS LGSYYGDWYFDLWGRGTLVT VSS (SEQ IDNO: 24).

[0162] In a specific embodiment, the V H The domain comprises SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, or the V H The VH domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications compared to SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, including amino acid substitutions, inversions, deletions, etc. The N-terminal and / or C-terminal sequences may have 1, 2, 3, 4, 5 or more amino acid truncations compared to SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. In a specific embodiment, the VH domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications compared to SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, including amino acid substitutions, inversions, deletions, etc. The N-terminal and / or C-terminal sequences may have 1, 2, 3, 4, 5 or more amino acid truncations compared to SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. H The domain comprises at least 100, 105, 110, 115, 116, 117, 118, 119, 120, 121, 122 or all of the consecutive amino acids of SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

[0163] Therefore, in a specific embodiment, any antibody used herein may comprise the following V L One of the sequences, or one or more of its modifications:

[0164] (1)QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMIYDVSYRPSGVSHRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTLV FGTGTKLTVL (SEQID NO: 25);

[0165] or

[0166] (3)NFMLTQPHSM SESPGKTVTI SCTRSSGSIA NNYVQWYQQR PGSSPTTVIYDDNQRPSGVPDRFSGSIDSS SNSASLTISG LKTEDEADYY CQSYDSSNVI FGGGTKLTVL G (SEQ ID NO: 27).

[0167] In a specific embodiment, the V L The domain comprises SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27, or the V L The domain is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical in sequence to SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27. Compared to SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27, V L The domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, including amino acid substitutions, inversions, deletions, etc. The N-terminal and / or C-terminal sequences may have 1, 2, 3, 4, 5 or more amino acid truncations compared to SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27. In a specific embodiment, V H The domain comprises at least 100, 105, 110, 115, 116, 117, 118, 119, 120, 121, 122 or all of the consecutive amino acids of SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

[0168] The scFv in various embodiments comprises any one or more of SEQ ID NOs: 4-9, 22-24, or 25-27. The scFv in specific embodiments comprises a sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more % identical to any one or more of SEQ ID NOs: 4-9, 22-24, or 25-27.

[0169] Various aspects of the present disclosure relate to antibodies or antigen-binding fragments comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of a heavy chain variable region from an antibody clone of B1, G7 or G9, and wherein the light chain variable region comprises LCDR1, LCDR2 and LCDR3 of a light chain variable region from the same corresponding clone of B1, G7 or G9. A further aspect relates to an antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a HCDR1, HCDR2 and HCDR3 having at least 80% sequence identity to, or having at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to, a heavy chain variable region from a B1, G7 or G9 antibody clone (or wherein 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any derivable range therein) sequence identity to the light chain variable region of the same corresponding clone of B1, G7 or G9.

[0170] Various aspects of the present disclosure relate to antibodies, antigen-binding fragments or polypeptides comprising a heavy chain variable region having HCDR1, HCDR2 and HCDR3, and a light chain variable region having LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 28 to 30, and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 31, 33 or "DVS".

[0171] Various aspects of the present disclosure relate to antibodies, antigen-binding fragments or polypeptides, which comprise a heavy chain variable region having HCDR1, HCDR2 and HCDR3, and a light chain variable region having LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 34 to 36, and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 37, 39 or "DAS".

[0172] Various aspects of the present disclosure relate to antibodies, antigen-binding fragments or polypeptides comprising a heavy chain variable region having HCDR1, HCDR2 and HCDR3, and a light chain variable region having LCDR1, LCDR2 and LCDR3, wherein HCDR1, HCDR2 and HCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 40 to 42, and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequence of SEQ ID NOs: 43, 45 or "DDN".

[0173] Polynucleotides encoding antibodies or portions thereof, antibody proteins, vectors comprising polynucleotides, vectors encoding antibody proteins, and cells comprising any of these agents are all included herein. In a specific embodiment, the antibody is used for any kind of chimeric protein. Polynucleotides encoding chimeric proteins, chimeric proteins, vectors comprising polynucleotides encoding chimeric proteins, and cells comprising any of these agents are all included herein. Chimeric proteins may or may not be chimeric receptors, chimeric antibodies, multispecific antibodies, labeled antibodies, antibody conjugates, etc. [A. Compositions based on TdT peptide antibodies]

[0174] The present disclosure relates at least to methods of treatment and / or detection using specific antibodies that target one or more TdT peptides and compositions utilizing the same. In various embodiments, the specific antibodies are used in one or more different compositions to target cancer cells that express the TdT peptide, and in specific embodiments, the peptide comprises ALYDKTKRI (SEQ ID NO: 3). In some embodiments, one of the compositions utilizing the antibody is cell-based, expressed in a non-natural receptor on the surface of the cell, and targets TdT. In some embodiments, one of the compositions utilizing the antibody is soluble and targets the TdT peptide outside the cell (although it can be delivered to an individual in the form of a cell, and the soluble protein comprising the antibody is subsequently secreted from the cell).

[0175] The present disclosure also relates to methods for identifying TdT peptides using any TdT peptide-based composition for any reason. Such methods may include detecting TdT peptides in a sample that needs to be processed. The sample may be from an individual who needs to determine the presence of TdT peptides for any reason. In a specific embodiment, the composition based on TdT peptide antibodies is used to detect cancer in a sample, such as the detection of cancer cells expressing TdT peptides in the sample, including one associated with the HLA-A2 complex in at least some cases. The TdT peptide may be associated with any HLA antigen, including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F or HLA-G.

[0176] In specific embodiments, the disclosure includes antibodies that bind a portion of a TdT protein, including binding a TdT peptide comprising, consisting of, or consisting essentially of SEQ ID NO: 3 or a variant thereof. Although the antibody can be of any kind, in specific embodiments, the antibody comprises a single chain variable fragment (scFv).

[0177] In various embodiments, the disclosure provides a variety of scFvs, which can be used alone or in any chimeric polypeptide, including any receptor and / or in any bispecific or multispecific antibody, including any BiTE, or in any conjugate. In a specific embodiment, the antibody comprises a sequence disclosed herein.

[0178] The scFv in various embodiments comprises any one or more of SEQ ID NOs: 4 to 9 or 22 to 27. The scFv in specific embodiments comprises a sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more % identical to any one or more of SEQ ID NOs: 4 to 9 or 22 to 27. In specific embodiments, the scFv comprises a complementarity determining region comprising a sequence identified by standard methods in the art.

[0179] Polynucleotides encoding antibodies or parts thereof, antibody proteins, vectors comprising polynucleotides, and vectors encoding antibody proteins are all included herein. In a specific embodiment, the antibody is used for any kind of chimeric protein. In a specific embodiment, the chimeric protein is a receptor, including a receptor that binds a TdT peptide, such as a receptor that contains an antibody that binds a TdT peptide. In a specific embodiment, the chimeric receptor is a chimeric T cell receptor (TCR) or a chimeric antigen receptor. Polynucleotides encoding chimeric TCRs comprising antibodies, chimeric TCR proteins, vectors comprising polynucleotides, and vectors encoding chimeric TCR proteins are all included herein. The antibody portion of a chimeric TCR can be configured in a variety of ways relative to TCR alpha and beta chains. Any vector can be used, including viral vectors and non-viral vectors. The viral vector can be an adenoviral vector, an adeno-associated viral vector, a retroviral vector, or a lentiviral vector. The non-viral vector can be a plasmid or a transposon.

[0180] In one embodiment, the chimeric receptor is a synthetic T cell receptor (TCR) and antigen receptor (STAR) (Liu et al., Sci Transl Med. 2021; 13(586):eabb5191), wherein the antigen receptor comprises the antigen recognition domain of an antibody and the constant region of a TCR molecule ( Figure 7 In various embodiments, the STAR receptor is a two-chain TCRαβ-based receptor having immunoglobulin heavy and light chain variable regions (V H and V L In a specific embodiment, the STAR receptor comprises V H and V L Fusion to the constant regions of the TCR α and β chains, respectively. Although a representative example of a STAR receptor comprises the B1 antibody described above and comprises two separate chains (B1 STAR), any antibody that binds to the TdT peptide can be used equally well in any STAR configuration.

[0181]

B1_STAR (2 independent chains)

[0182]

B1_STAR VH-TCRA polynucleotide (VH of B1 antibody linked to TCRα chain H domain)

[0183] ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG

[0184] CCGGCTCAAG TTCAGCTGGT GCAGAGCGGA GCGGAGGTGA AGAAGCCAGG GTCTTCCGTT

[0185] AAGGTCTCTT GTAAGGCCTC AGGCGGAACT TTCTCATCTT ACGCTATTTC CTGGGTAAGG

[0186] CAAGCACCAG GACAGGGATT AGAATGGATG GGGGGGATTA TCCCCATTTT CGGTACCGCG

[0187] AACTACGCCC AAAAATTTCA AGGTCGGGTA ACCATCACCG CCGATGAATC GACATCTACC

[0188] GCCTACATGG AACTGAGCTC TCTTCGGAGC GAGGACACCG CAGTCTATTA CTGCGCGAGA

[0189] GACGGGTATA GTGGTAGCTA CTACTATTAT TACGGCATGG ACGTTTGGGG CCAGGGGACC

[0190] TTAGTGACGG TGAGCAGCGG ATCCCCATAC ATTCAAAATC CAGAACCTGC GGTATACCAA

[0191] TTGAAAGACC CAAGAAGCCA GGATTCCACG CTGTGTCTGT TTACAGACTT CGACTCACAG

[0192] ATCAATGTTC CCAAGACAAT GGAAAGCGGG ACGTTCATCA CCGATAAATG CGTTCTGGAC

[0193] ATGAAGGCTA TGGATTCTAA AAGTAATGGT GCTATAGCCT GGAGTAACCA GACCTCATTT

[0194] ACTTGCCAAG ACATTTTCAA GGAGACCAAT GCTACATATC CATCCTCCGA CGTCCCTTGT

[0195] GACGCCACTC TGACCGAGAA ATCTTTTGAG ACCGATATGA ACCTGAACTT TCAGAACCTA

[0196] CTGGTTATCG TTCTGAGAAT ACTGTTGCTC AAAGTCGCCG GATTCAACCTTCTCATGACTCTGAGACTGT GGAGTAGC (SEQ ID NO: 10)

[0197]

B1_STAR VH-TCRA peptide (VH of B1 antibody linked to TCRα chain H domain)

[0198] MALPVTALLL PLALLLHAAR PAQVQLVQSG AEVKKPGSSV KVSCKASGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTA NYAQKFQGRV TITADESTST AYMELSSLRSEDTAVYYCARDGYSGSYYYY YGMDVWGQGT LVTVSSGSPY IQNPEPAVYQ LKDPRSQDSTLCLFTDFDSQINVPKTMESG TFITDKCVLD MKAMDSKSNG AIAWSNQTSF TCQDIFKETNATYPSSDVPCDATLTEKSFE TDMNLNFQNL LVIVLRILLL KVAGFNLLMT LRLWSS (SEQ ID NO: 11)

[0199]

B1_STAR VL-TCRB polynucleotide (VL of B1 antibody linked to TCRβ chain) L domain)

[0200] ATGGAGACAG ACACACTCCT Gctatgggtg ctgctgctct GGGTTCCAGGTTCCACAGGTCAGTCTGCTC TGACCCAACC TGCCAGCGTC TCCGGCAGTC CAGGACAAAGTATCACCATCAGCTGCACTG GGACTTCCAG CGATGTGGGG GGCTACAATCATTCAGCCGACCGAGCCG AGGCCCCCAA GCTGATGATT TATGATGTCT CATACAGACCGAGCGGAGTGAGCCACCGTT TTAGTGGCTC CAAGTCCGGA AACACCGCTT CACTAACAATCAGTGGCTTACAAGCAGAGG ACGAAGCCGA TTATTACTGC TCATCTTACA CCAGTTCCAGTACTTGTGTGTGACTGGACTGACTGACT GGCACGCGTG AGGACTTGAGGAATGTGACGCCCACCGAAGG TCTCTTTGTT TGAGCCGTCT AAGGCAGAAA TTGCTAATAAACAAAAGGCCACCCTAGTGT GTCTCGCGAG GGGATTCTTT CCTGATCACG TTGAGTTGTCCTGGTGGGTGAATGGTAAGG AGGGTTCCCTAG AGGCATACAAGGAGTCGAACTACTCCTACT GCCTGTCATC TAGACTCCGA GTCAGTGCCA CGTTCTGGCATAACCCCCGTAACCATTTTA GGTGCCAGGT GCAATTCCAC GGGCTTAGTG AAGAGGACAAGTGGCCAGAGGGGTCCCCAA AGCCGGTGAC CCAAAATAGAGTCTC CTTGGGGGGCGCGCCGATTGTGGGATTACCA GCGCGAGCTA TCAGCAGGGA GTCCTGAGTG CAACGATACTCTACGAGATATTACTGGGAA AGGCTACATT ATATGCAGTG TTGGTGAGCA CTCTGGTCGTCATGGCAATGGTTAAGAGAA AGAATAGCTA A(SEQTAGCTA) ID NO.

[0201]

B1_STAR VL-TCRB peptide (VL domain of B1 antibody linked to TCRβ chain)

[0202] METDTLLLWV LLLWVPGSTG QSALTQPASV SGSPGQSITI SCTGTSSDVGGYNYVSWYQQHPGKAPKLMI YDVSYRPSGV SHRFSGSKSG NTASLTISGL QAEDEADYYCSSYTSSSTLVFGTGTKLTVL GTREDLRNVT PPKVSLFEPS KAEIANKQKA TLVCLARGFFPDHVELSWWVNGKEVHSGVC TDPQAYKESN YSYCLSSRLR VSATFWHNPR NHFRCQVQFHGLSEEDKWPEGSPKPVTQNI SAEAWGRADC GITSASYQQG VLSATILYEI LLGKATLYAVLVSTLVVMAMVKRKNS(SEQ ID NO: 13)

[0203] In one embodiment, the chimeric receptor is a chimeric T cell receptor (cTCR). In this case, the scFv is fused to the TCR alpha and / or TCR beta chain. In a specific embodiment, both the VH and VL domains are linked to the TCR alpha chain or the TCR beta chain. Although a representative example of a cTCR includes the B1 antibody (B1 cTCR) described above linked to the TCR alpha or beta chain, any antibody that binds to the TdT peptide can be used with any configuration of the TCR alpha beta chain. L and V H If all variable domains are attached to the TCRα chain, the TCRβ chain will be considered “empty” (e.g., lacking variable domain sequences). L and V H domains are attached to the TCRβ chain, the TCRα chain would be considered “empty” (e.g., lacking the variable domain sequence).

[0204]

B1.cTCR

[0205]

B1_cTCR VHVL-TCRA polynucleotide (V of B1 antibody linked to TCRα chain L and V H domain)

[0206] ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCACGCCGCCAGGCCGGCTCAAG TTCAGCTGGT GCAGAGCGGA GCGGAGGTGA AGAAGCCAGGGTCTTCCGTTAAGGTCTCTT GTAAGGCCTC AGGCGGAACT TTCTCATCTT ACGCTATTTCCTGGGTAAGGCAAGCACCAG GACAGGGATT AGAATGGATG GGGGGGATTA TCCCCATTTTCGGTACCGCGAACTACGCCC AAAAATTTCA AGGTCGGGTA ACCATCACCG CCGATGAATCGACATCTACCGCCTACATGG AACTGAGCTC TCTTCGGAGC GAGGACACCG CAGTCTATTACTGCGCGAGAGACGGGTATA GTGGTAGCTA CTACTATTAT TACGGCATGG ACGTTTGGGGCCAGGGGACCTTAGTGACGG TGAGCAGCGG AGGAGGCGGC GGGTCCCAGT CTGCTCTGACCCAACCTGCCAGCGTCTCCG GCAGTCCAGG ACAAAGTATC ACCATCAGCT GCACTGGGACTTCCAGCGATGTGGGGGGCT ACAATTACGT AAGTTGGTAC CAGCAACACC CTGGGAAGGCCCCCAAGCTGATGATTTATG ATGTCTCATA CAGACCGAGC GGAGTGAGCC ACCGTTTTAGTGGCTCCAAGTCCGGAAACA CCGCTTCACT AACAATCAGT GGCTTACAAG CAGAGGACGAAGCCGATTATTACTGCTCAT CTTACACCAG TTCCAGTACG CTTGTGTTTG GTACTGGAACAAAGTTGACTGTGCTGGGCG GATCCCCATA CATTCAAAAT CCAGAACCTG CGGTATACCAATTGAAAGACCCAAGAAGCC AGGATTCCAC GCTGTGTCTG TTTACAGACT TCGACTCACAGATCAATGTTCCCAAGACAA TGGAAAGCGG GACGTTCATCACCGATAAAT GCGTTCTGGACATGAAGGCTATGGATTCTA AAAGTAATGG TGCTATAGCC TGGAGTAACC AGACCTCATTTACTTGCCAAGACATTTTCA AGGAGACCAA TGCTACATAT CCATCCTCCG ACGTCCCTTGTGACGCCACTCTGACCGAGA AATCTTTTGA GACCGATATG AACCTGAACT TTCAGAACCTACTGGTTATCGTTCTGAGAA TACTGTTGCT CAAAGTCGCCGGATTCAACCTTCTCATGACTCTGAGACTGTGGAGTAGC (SEQ ID NO: 14)

[0207]

B1_cTCR VHVL-TCRA polypeptide (V of B1 antibody linked to TCRα chain L and V H domain)

[0208] MALPVTALLL PLALLLHAAR PAQVQLVQSG AEVKKPGSSV KVSCKASGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTA NYAQKFQGRV TITADESTST AYMELSSLRSEDTAVYYCARDGYSGSYYYY YGMDVWGQGT LVTVSSGGGG GSQSALTQPA SVSGSPGQSITISCTGTSSDVGGYNYVSWY QQHPGKAPKL MIYDVSYRPS GVSHRFSGSK SGNTASLTISGLQAEDEADYYCSSYTSSST LVFGTGTKLT VLGGSPYIQN PEPAVYQLKD PRSQDSTLCLFTDFDSQINVPKTMESGTFI TDKCVLDMKA MDSKSNGAIA WSNQTSFTCQ DIFKETNATYPSSDVPCDATLTEKSFETDM NLNFQNLLVI VLRILLLKVA GFNLLMTLRL WSS (SEQ ID NO: 15)

[0209]

B1_cTCR VHVL-TCRB polynucleotide (V of B1 antibody linked to TCRβ chain L and V H domain)

[0210] ATGGCCTTAC CAGTGACCGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCACGCCGCCAGGCCGGCTCAAG TTCAGCTGGT GCAGAGCGGA GCGGAGGTGA AGAAGCCAGGGTCTTCCGTTAAGGTCTCTT GTAAGGCCTC AGGCGGAACT TTCTCATCTT ACGCTATTTCCTGGGTAAGGCAAGCACCAG GACAGGGATT AGAATGGATG GGGGGGATTA TCCCCATTTTCGGTACCGCGAACTACGCCC AAAAATTTCA AGGTCGGGTA ACCATCACCG CCGATGAATCGACATCTACCGCCTACATGG AACTGAGCTC TCTTCGGAGC GAGGACACCG CAGTCTATTACTGCGCGAGAGACGGGTATA GTGGTAGCTA CTACTATTAT TACGGCATGG ACGTTTGGGGCCAGGGGACCTTAGTGACGG TGAGCAGCGG AGGAGGCGGC GGGTCCCAGT CTGCTCTGACCCAACCTGCCAGCGTCTCCG GCAGTCCAGG ACAAAGTATC ACCATCAGCT GCACTGGGACTTCCAGCGATGTGGGGGGCT ACAATTACGT AAGTTGGTAC CAGCAACACC CTGGGAAGGCCCCCAAGCTGATGATTTATG ATGTCTCATA CAGACCGAGC GGAGTGAGCC ACCGTTTTAGTGGCTCCAAGTCCGGAAACA CCGCTTCACT AACAATCAGT GGCTTACAAG CAGAGGACGAAGCCGATTATTACTGCTCAT CTTACACCAG TTCCAGTACG CTTGTGTTTG GTACTGGAACAAAGTTGACTGTGCTGGGCG GATCCACGCG TGAGGACTTG AGGAATGTGA CGCCACCGAAGGTCTCTTTGTTTGAGCCGT CTAAGGCAGA AATTGCTAAT AAACAAAAGG CCACCCTAGTGTGTCTCGCGAGGGGATTCT TTCCTGATCA CGTTGAGTTGTCCTGGTGGG TGAATGGTAAGGAGGTTCATAGCGGTGTCT GCACCGACCC CCAGGCATAC AAGGAGTCGA ACTACTCCTACTGCCTGTCATCTAGACTCC GAGTCAGTGC CACGTTCTGG CATAACCCCC GTAACCATTTTAGGTGCCAGGTGCAATTCC ACGGGCTTAG TGAAGAGGAC AAGTGGCCAG AGGGGTCCCCAAAGCCGGTGACCCAAAATA TCTCTGCGGA AGCTTGGGGG CGCGCCGATT GTGGGATTACCAGCGCGAGCTATCAGCAGG GAGTCCTGAG TGCAACGATA CTCTACGAGA TATTACTGGGAAAGGCTACATTATATGCAG TGTTGGTGAG CACTCTGGTC GTCATGGCAA TGGTTAAGAG AAAGAATAGCTAA(SEQ ID NO:16)

[0211]

B1_cTCR VHVL-TCRB polypeptide (V L and V H domains of B1 antibody linked to TCRβ chain)

[0212] MALPVTALLL PLALLLHAAR PAQVQLVQSG AEVKKPGSSV KVSCKASGGTFSSYAISWVRQAPGQGLEWM GGIIPIFGTA NYAQKFQGRV TITADESTST AYMELSSLRSEDTAVYYCARDGYSGSYYYY YGMDVWGQGT LVTVSSGGGG GSQSALTQPA SVSGSPGQSITISCTGTSSDVGGYNYVSWY QQHPGKAPKL MIYDVSYRPS GVSHRFSGSK SGNTASLTISGLQAEDEADYYCSSYTSSST LVFGTGTKLT VLGGSTREDL RNVTPPKVSL FEPSKAEIANKQKATLVCLARGFFPDHVEL SWWVNGKEVH SGVCTDPQAY KESNYSYCLS SRLRVSATFWHNPRNHFRCQVQFHGLSEED KWPEGSPKPV TQNISAEAWG RADCGITSAS YQQGVLSATILYEILLGKATLYAVLVSTLV VMAMVKRKNS (SEQ ID: 17).

[0213] An example of a TCRβ chain that is considered “empty” (lacking any antibody material) is as follows, and can be combined in a cell with a TCRα chain (itself combined with V H and V L Domain connection) combination:

[0214]

TCRβ_Empty Polynucleotide

[0215] ATGGAGACAG ACACACTCCT Gctatgggtg ctgctgctct gggttCCAGGTTCCACAGGTGGCGGCGGTG GGTCAGAACA AAAATTGATT TCAGAGGAAG ATCTCGGAGGGGGAGGGTCAACGCGTGAGG ACTTGAGGAA TGTGACGCCA CCGAAGGTCT CTTTGTTTGAGCCGTCTAAGGCAGAAATTG CTAATAAACA AAAGGCCACC CTAGTGTGTC TCGCGAGGGGATTCTTTCCTGATCACGTTG AGTTGTCCTG GTGGGTGAAT GGTAAGGAGG TTCATAGCGGTGTCTGCACCGACCCCCAGG CATACAAGGA GTCGAACTAC TCCTACTGCC TGTCATCTAGACTCCGAGTCAGTGCCACGT TCTGGCATAA CCCCCGTAAC CATTTTAGGT GCCAGGTGCAATTCCACGGGCTTAGTGAAG AGGACAAGTG GCCAGAGGGG TCCCCAAAGC CGGTGACCCAAAATATCTCTGCGGAAGCTT GGGGGCGCGC CGATTGTGGG ATTACCAGCG CGAGCTATCAGCAGGGAGTCCTGAGTGCAA CGATACTCTA CGAGATATTA CTGGGAAAGG CTACATTATATGCAGTGTTGGTGAGCACTC TGGTCGTCAT GGCAATGGTT AAGAGAAAGA ATAGCTAA(SEQ ID NO:20)

[0216]

TCRβ_empty polypeptide

[0217] METDTLLLWV LLLWVPGSTG GGGGSEQKLI SEEDLGGGGS TREDLRNVTPPKVSLFEPSKAEIANKQKAT LVCLARGFFP DHVELSWWVN GKEVHSGVCT DPQAYKESNYSYCLSSRLRVSATFWHNPRN HFRCQVQFHG LSEEDKWPEG SPKPVTQNIS AEAWGRADCGITSASYQQGVLSATILYEIL LGKATLYAVL VSTLVVMAMV KRKNS (SEQ ID NO: 21)

[0218] Representative sequences of TCR alpha chains that are "empty" (lacking any antibody material) are known in the art.

[0219] In one embodiment, there are methods and compositions utilizing bispecific T cell engagers of specific sequences comprising any antibody that binds to a TdT peptide. Although the specific example of an engager uses the B1 antibody clone described above, in other embodiments, any TdT specific antibody is used for the BiTE.

[0220]

TdT B1 BiTE polynucleotide (B1 antibody linked to anti-CD3 antibody)

[0221] ATGGATTGGA TCTGGCGCAT CCTGTTTCTC GTGGGAGCCG CCACAGGCGCCCATTCTGCTCAAGTTCAGC TGGTGCAGAG CGGAGCGGAG GTGAAGAAGC CAGGGTCTTCCGTTAAGGTCTCTTGTAAGG CCTCAGGCGG AACTTTCTCA TCTTACGCTA TTTCCTGGGTAAGGCAAGCACCAGGACAGG GATTAGAATG GATGGGGGGG ATTATCCCCA TTTTCGGTACCGCGAACTACGCCCAAAAAT TTCAAGGTCG GGTAACCATC ACCGCCGATG AATCGACATCTACCGCCTACATGGAACTGA GCTCTCTTCG GAGCGAGGAC ACCGCAGTCT ATTACTGCGCGAGAGACGGGTATAGTGGTA GCTACTACTA TTATTACGGC ATGGACGTTT GGGGCCAGGGGACCTTAGTGACGGTGAGCA GCGGAGGAGG CGGCGGGTCC CAGTCTGCTC TGACCCAACCTGCCAGCGTTCTCCGGCAGTC CAGGACAAAG TATCACCATC AGCTGCACTG GGACTTCCAGCGATGTGGGGGGCTACAATT ACGTAAGTTG GTACCAGCAA CACCCTGGGA AGGCCCCCAAGCTGATGATTTATGATGTCT CATACAGACC GAGCGGAGTG AGCCACCGTT TTAGTGGCTCCAAGTCCGGAAACACCGCTT CACTAACAAT CAGTGGCTTA CAAGCAGAGG ACGAAGCCGATTATTACTGCTCATCTTACA CCAGTTCCAG TACGCTTGTG TTTGGTACTG GAACAAAGTTGACTGTGCTGGGCTCCGGAG GTGGTGGATC CGATATCAAA CTGCAGCAGT CAGGGGCTGAACTGGCAAGACCTGGGGCCT CAGTGAAGAT GTCCTGCAAG ACTTCTGGCT ACACCTTTACTAGGTACACGATGCACTGGG TAAAACAGAG GCCTTGGACAGGGTCTGGAAT GGATTGGATACATTAATCCTAGCCGTGGTT ATACTAATTA CAATCAGAAG TTCAAGGACA AGGCCACATTGACTACAGACAAATCCTCCA GCACAGCCTA CATGCAACTG AGCAGCCTGA CATCTGAGGACTCTGCAGTCTATTACTGTG CAAGATATTA TGATGATCAT TACTGCCTTG ACTACTGGGGCCAAGGCACCACTCTCACAG TCTCCTCAGG TGGTGGTGGT TCTGGCGGCG GCGGCTCCGGTGGTGGTGGTTCTGACATTC AGCTGACCCA GTCTCCAGCA ATCATGTCTG CATCTCCAGGGGAGAAGGTCACCATGACCT GCAGAGCCAG TTCAAGTGTA AGTTACATGA ACTGGTACCAGCAGAAGTCAGGCACCTCCC CCAAAAGATG GATTTATGAC ACATCCAAAG TGGCTTCTGGAGTCCCTTATCGCTTCAGTG GCAGTGGGTC TGGGACCTCA TACTCTCTCA CAATCAGCAGCATGGAGGCTGAAGATGCTG CCACTTATTA CTGCCAACAG TGGAGTAGTA ACCCGCTCACGTTCGGTGCTGGGACCAAGC TGGAGCTGAA ATCCTAA(SEQ ID NO:18)

[0222]

TdT B1 BiTE polypeptide (B1 antibody linked to anti-CD3 antibody)

[0223] MDWIWRILFL VGAATGAHSA QVQLVQSGAE VKKPGSSVKV SCKASGGTFSSYAISWVRQAPGQGLEWMGG IIPIFGTANY ​​AQKFQGRVTI TADESTSTAY MELSSLRSEDTAVYYCARDGYSGSYYYYYG MDVWGQGTLV TVSSGGGGGS QSALTQPASV SGSPGQSITISCTGTSSDVGGYNYVSWYQQ HPGKAPKLMI YDVSYRPSGV SHRFSGSKSG NTASLTISGLQAEDEADYYCSSYTSSSTLV FGTGTKLTVL GSGGGGSDIK LQQSGAELAR PGASVKMSCKTSGYTFTRYTMHWVKQRPGQ GLEWIGYINP SRGYTNYNQK FKDKATLTTD KSSSTAYMQLSSLTSEDSAVYYCARYYDDH YCLDYWGQGT TLTVSSGGGG SGGGGSGGGG SDIQLTQSPAIMSASPGEKVTMTCRASSSV SYMNWYQQKS GTSPKRWIYD TSKVASGVPY RFSGSGSGTSYSLTISSMEAEDAATYYCQQ WSSNPLTFGA GTKLELKS(SEQ ID NO: 19)

[0224] In various embodiments, any polynucleotide comprising a sequence encoding a chimeric receptor protein (a chimeric TCR or STAR or a chimeric antigen receptor) may also encode one or more other gene products. In a specific embodiment, a polynucleotide encoding a chimeric receptor protein may also encode another gene product that also uses an antibody (including an anti-TdT peptide antibody). In a specific embodiment, the polynucleotide encoding the chimeric receptor protein also includes a sequence encoding a bispecific T cell engager (BiTE), including a sequence comprising an anti-TdT peptide antibody. In certain cases, the polynucleotide encoding the TCR protein is a different polynucleotide encoding a BiTE, and the BiTE comprises an antibody specific for TdT or a TdT peptide.

[0225] In a specific embodiment, the chimeric receptor used may include a chimeric antigen receptor (CAR). CAR can be first generation, second generation, third generation, and so on. In a specific embodiment, the antibody portion of the CAR targets a TdT peptide that may or may not be associated with HLA. The hinge, transmembrane domain, CD3ζ (or a domain with similar function), and optional one or more costimulatory domains can be of any suitable type.

[0226] In a specific embodiment, the present disclosure relates to TdT-specific cytotoxic receptors and methods of using the same. The present disclosure includes novel agents that recognize and eliminate any type of cancer cells (including blood cancers, such as acute leukemia cells) without causing substantial damage to normal cells. The disclosure includes T cell-based (chimeric T cell receptors (TCRs)) and / or recombinant protein-based (bispecific T cell engagers) agents to target specific TdT peptides, including peptides present on the cell surface, for example in the context of HLA-A2. The potent activity of TdT-redirected T cells in preclinical models of human acute leukemia is demonstrated herein.

[0227] This article contains enhanced sensitivity of tumor antigen targeting through chimeric receptors. The active development of CAR- and TCR-based immune cell therapies and their clinical evaluation have prompted the study of mechanisms that regulate tumor-specific receptor sensitivity and efficacy. Although most CARs target antigens that are abundantly expressed on tumor cells, it is important to understand the limits of their antigen sensitivity and design new strategies to overcome low responsiveness to tumors and reduced target antigen expression. Previous studies have shown that CARs have significantly reduced sensitivity compared to TCRs and tend to "ignore" tumor variants with reduced antigen surface density (Watanabe et al., 2015; Gudipati et al., 2020). To overcome this limitation, the inventors combined high TCR sensitivity with the versatility of CAR-based targeting to develop a highly sensitive chimeric TCR and antibody-derived antigen recognition moiety.

[0228] Embodiments of the present disclosure allow for redirection of CD4+ and CD8+ T cells to tumor antigens presented by class I MHC. T cells expressing natural TdT-specific TCRs are deleted during thymic selection, thereby excluding the selection or expansion of natural TdT-specific T cells in patients who need them (e.g., leukemia). To overcome this limitation, the inventors have identified an antibody binder that specifically recognizes dominant TdT peptides in the context of HLA-A02, which is the most common class I MHC allele in the Western world. Since this binding does not require CD8 co-receptors, the expression of TdT-specific cTCRs can effectively redirect CD8+ and CD4+ T cells to elicit effector functions against TdT+ leukemias, which is different from conventional MHC I-specific TCRs that only activate CD8+ T cells. According to current preclinical and clinical evidence, CD4+ T cell activation improves TdT-specific T cell responses and overall anti-leukemia activity in specific embodiments.

[0229] As shown elsewhere in this article, T cells expressing TdT-specific cTCRs have more potent anti-leukemic activity compared to T cells expressing similar TdT-specific chimeric antigen receptors (CARs). These results suggest that, in various embodiments, optimal recognition of rare TdT / HLA complexes requires highly sensitive TCR signaling. As shown herein, unmodified T cells can be effectively redirected to fight TdT+ leukemias in the presence of soluble TdT-specific T cell engagers, which similarly activate TCR signaling in bystander, unmodified T cells. These results provide a scientific rationale for the development of cellular and non-cellular strategies to target TdT using cTCR transgenic T cells or recombinant bispecific engagers, respectively. Compared to autologous engineered T cells, bispecific engagers offer ready availability and reduced manufacturing costs, and their use during treatment, including early in the course of treatment, may enhance the anti-tumor activity of standard of care treatments, particularly in high-risk T-ALL and AML patients ( Figure 2 ). Thus, in specific embodiments of the present disclosure, there are methods and compositions for employing adaptive T cell therapy and bispecific T cell engagers on the same individual.

[0230] In various embodiments, methods and compositions are directed to a recombinant T cell receptor (TCR) comprising a single chain variable fragment (scFv) specific for terminal deoxynucleotidyl transferase (TdT) or a TdT peptide. Any embodiment associated with the receptor may be used, including non-natural TCR polypeptides, recombinant polynucleotides encoding TCR polypeptides, cells comprising TCR polypeptides (including those isolated from nature, obtained commercially, obtained from donors or individuals treated with them, etc.) or polynucleotides encoding TCR polypeptides, any type of vector polynucleotide expressing TCR polypeptides, cells containing one or more recombinant polynucleotides, and so on. In a specific embodiment of the recombinant TCR, the TCR comprises a binder targeting a TdT peptide, such as an antibody, including a peptide that may be associated with HLA-A02 on the surface of a cell (including the surface of a cancer cell, not a non-cancerous cell).

[0231] The TCR is non-natural in that it has been designed by human hands to comprise an antibody that binds to the TdT peptide and in some embodiments structurally comprises a disulfide-linked membrane-anchored heterodimeric protein comprising an alpha chain and a beta chain. In a specific embodiment, the TCR alpha chain and / or the TCR beta chain in the recombinant TCR have one or more manual modifications, such as to prevent cross-pairing with endogenous TCRs present in the cell and / or to enhance heterologous pairing with each other. In a specific embodiment, the alpha chain and / or the beta chain is not from a human, but from another mammal, such as a mouse, rat, or monkey. In a specific embodiment, the V of the anti-TdT peptide antibody L and VH are all connected to the same TCR chain (α or β) ( Figure 7 In some embodiments, both TCR chains are linked to the V H or V L In a specific embodiment, representative sequences can be used in the compositions of the present disclosure, wherein the bold underlined sequences have been modified compared to the wild type:

[0232]

TCRα

[0233]

[0234]

TCRβ

[0235]

[0236] Any protein, polynucleotide and / or cell included herein may be included in the composition. The cell in the composition may be of any kind, including immune cells, such as immune effector cells. The cell in the composition may be a T cell, a NK cell, a NK T cell, a B cell, a macrophage, a neutrophil or a combination thereof. When the cell is a T cell, the T cell may be an αβT cell, a γδT cell, a T helper cell, an invariant natural killer T (iNKT) cell, a cytotoxic T cell, a T regulatory cell natural killer (NK) cell, or a combination thereof. In various embodiments, a cell may comprise (a) a polynucleotide comprising (1) a sequence encoding a recombinant TCR comprising a scFv specific for TdT or a TdT peptide; (2) a sequence encoding a BiTE comprising an antibody specific for TdT or a TdT peptide; or (b) a first polynucleotide comprising a sequence encoding a recombinant TCR comprising a scFv specific for TdT or a TdT peptide; and a second polynucleotide comprising a sequence encoding a BiTE comprising an antibody specific for TdT or a TdT peptide.

[0237] B. Antibody Types

[0238] The TdT-specific antibodies can be of any type. Antibodies can be whole immunoglobulins, chimeric antibodies or hybrid antibodies of any isotype or classification that are specific for two or more antigens. They can also be fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.), including hybrid fragments. Immunoglobulins also include natural, synthetic or genetically engineered proteins that act as antibodies by binding to specific antigens to form complexes. The term antibody includes transgenic or otherwise modified forms of immunoglobulins.

[0239] The term "monomer" refers to an antibody that contains only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody that contains two Ig units, which are interconnected by the constant domain (Fc, or crystallizable fragment, region) of the antibody heavy chain. The complex can be stabilized by connecting (J) chain proteins. The term "multimer" refers to an antibody that contains more than two Ig units, which are interconnected by the constant domain (Fc region) of the antibody heavy chain. The complex can be stabilized by connecting (J) chain proteins.

[0240] The term "bivalent antibody" refers to an antibody that contains two antigen-binding sites. The two binding sites may have the same antigen-specificity or may be bispecific, meaning that the two antigen-binding sites have different antigen-specificities.

[0241] Bispecific antibodies are a class of antibodies with two complements, which have different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be double complements, wherein bispecific antibodies can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed by a pair of different single-domain antibodies called "nanoantibodies". Single-domain antibodies are derived from cartilaginous fish and camelids and modified. Nanoantibodies can be connected together using typical techniques of those skilled in the art through joints; This method of selecting and connecting nanoantibodies is described in PCT Publication Nos. WO2015044386A1, WO2010037838A2, and Bever et al., Anal Chem. 86: 7875-7882 (2014), each of which is incorporated herein by reference as a whole.

[0242] Bispecific antibodies can be constructed as: full IgG, Fab'2, Fab'PEG, diabodies, or alternatively as scFv. Diabodies and scFvs can be constructed without an Fc region, using only the variable domains, which may reduce the impact of anti-idiotypic responses. Bispecific antibodies can be produced by a variety of methods, including but not limited to fusion of hybridomas or connection of Fab' fragments. For example, see Songsivilai and Lachmann, Clin. Exp. Immunol. 79: 315-321 (1990); Kostelny et al., J. Immunol. 148: 1547-1553 (1992), each of which is incorporated herein by reference in its entirety.

[0243] In certain aspects, the antigen binding domain can be multispecific or heterospecific, by combining V domains that bind different antigens. H and V LFor example, an antibody may bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Thus, aspects may include, but are not limited to, bispecific, trispecific, tetraspecific and other multispecific antibodies or antigen-binding fragments thereof, to epitopes and other targets, such as Fc receptors on effector cells.

[0244] In some embodiments, multispecific antibodies can be used and directly linked via short flexible polypeptide chains using conventional methods known in the art. One such example is diabodies, which are bivalent, bispecific antibodies in which V H and V L The domains are expressed on a single polypeptide chain, and a linker that is too short does not allow pairing between domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain, thereby generating two antigen binding sites. The linker function is applicable to embodiments of tri-antibodies, tetra-antibodies, and high-order antibody multimers (e.g., see Hollinger et al., Proc Natl. Acad. Sci. USA 90: 6444-6448 (1993); Polijaket al., Structure 2: 1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248: 47-66 (2001)).

[0245] Bispecific antibodies, in contrast to bispecific full antibodies, may also be advantageous because they can be easily constructed and expressed in E. coli. It is easy to select bispecific antibodies (and other polypeptides, such as antibody fragments) with appropriate binding specificity using phage display (WO94 / 13804) from libraries. If one arm of a bispecific antibody remains unchanged, for example, with specificity for a protein, a library can be established where the other arm changes, and antibodies with appropriate specificity can be selected. Bispecific full antibodies can be manufactured by substitution engineering methods described in Ridgeway et al., (Protein Eng., 9: 616-621, 1996) and Krah et al., (N Biotechnol. 39: 167-173, 2017), which are hereby incorporated by reference in their entirety.

[0246] Heteroconjugate antibodies consist of two covalently linked monoclonal antibodies of different specificities. See, e.g., U.S. Pat. No. 6,010,902, herein incorporated by reference in its entirety.

[0247] The part of the Fv fragment of an antibody molecule that binds to an antigenic epitope with high specificity is referred to herein as "patch". The patch is composed of amino acid residues that contact the antigenic epitope to promote antigen recognition. Each of the two Fv fragments of an antibody is composed of two variable domains VH and VL in a dimeric configuration. The primary structure of each variable domain includes three hypervariable loops, which are separated by a framework region (FR) and flanked by framework regions (FR). The hypervariable loop is the region with the highest primary sequence variability in the antibody molecule from any mammal. The term hypervariable loop is sometimes used interchangeably with the term "complementarity determining region (CDR)". The length of the hypervariable loop (or CDR) varies from antibody molecule to antibody molecule. The framework region of all antibody molecules from a given mammal has a very high primary sequence similarity / consistency. A person skilled in the art can use the consensus of the framework region to identify the framework region and the hypervariable loops (or CDRs) interspersed between the framework regions. The hypervariable loops are given identification names to distinguish their positions in the polypeptide and in which domain they appear. The CDRs in the VL domain are identified as L1, L2, and L3, with L1 occurring most distally and L3 occurring closest to the CL domain. CDRs may also be designated as CDR-L1, CDR-L2, and CDR-L3. L3 (CDR-L3) is generally the region with the highest variability among all antibody molecules produced by a given organism. CDRs are regions of the polypeptide chain that are linearly arranged in the primary structure and are separated from each other by framework regions. The amino terminus (N-terminus) of the VL chain is designated as FR1. The region identified as FR2 occurs between the L1 and L2 hypervariable loops. FR3 occurs between the L2 and L3 hypervariable loops, with the FR4 region being closest to the CL domain. This structure and nomenclature is repeated in the VH chain, which includes three CDRs identified as CDR-H1, CDR-H2, and CDR-H3. Most of the amino acid residues in the variable domains or Fv fragments (VH and VL) are part of the framework regions (approximately 85%). The three-dimensional or tertiary structure of an antibody molecule is such that the framework regions are located further inside the molecule and provide the majority of the structure, while the CDRs are located on the outer surface of the molecule.

[0248] Several methods have been developed and can be used by those skilled in the art to identify the exact amino acids that make up each of these regions. This can be accomplished using any of a variety of sequence alignment methods and algorithms that can identify the conserved amino acid residues that make up the framework regions, thereby identifying CDRs that may vary in length but are located between framework regions. Three commonly used methods have been developed to identify the CDRs of antibodies: Kabat (as described in TT Wu and EA Kabat, "AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATION FOR ANTIBODY COMPLEMENTARITY," J Exp Med, vol. 132, no. 2, pp. 211-250, August 1970); Chothia (as described in C. Chothia et al., "Conformations of immunoglobulin hypervariable regions," Nature, vol. 342, no. 6252, pp. 877-883, December 1989); and IMGT (as described in M.-P. Lefranc et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, January 2003). These methods all include a unique numbering system for identifying the amino acid residues that make up the variable region. In most antibody molecules, the amino acid residues that actually contact the antigen epitope appear in the CDR, although in some cases, residues within the framework region contribute to antigen binding.

[0249] One skilled in the art can use any of several methods to determine the position of an antibody. These methods include:

[0250] (1) Based on the chemical properties of the amino acid sequence of the antibody variable region and the composition of the epitope, the tertiary structure of the antibody / epitope binding interaction is predicted in silico.

[0251] (2) Hydrogen-deuterium exchange and mass spectrometry.

[0252] (3) Peptide fragmentation and peptide mapping methods, one of which generates multiple overlapping peptide fragments from the entire length of the polypeptide and evaluates the binding affinity of these peptides for the epitope.

[0253] (4) Analysis of antibody phage display libraries, in which genes encoding mammalian antibody Fab fragments are expressed by phage in a manner incorporated into the phage coat. The Fab-expressing phage population is then allowed to interact with an antigen that has been fixed or may be expressed by a different exogenous expression system. Non-binding Fab fragments are washed away, leaving only the specific binding Fab fragments attached to the antigen. The bound Fab fragments can be easily isolated and the genes encoding them determined. This method can also be used for smaller regions of Fab fragments, including Fv fragments or specific VH and VL domains (as appropriate).

[0254] In some aspects, affinity-matured antibodies are enhanced by one or more modifications in one or more CDRs thereof, which results in an improved affinity of the antibody to the target antigen compared to a parent antibody without these changes. Certain affinity-matured antibodies have nanomolar or picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art, for example, Marks et al., Bio / Technology 10: 779 (1992) describes affinity maturation by mixing VH and VL domains, and random mutagenesis of CDRs and / or framework residues used in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525: 309-22 (2009), combined with computational methods demonstrated in Tiller et al., Front. Immunol. 8: 986 (2017).

[0255] Chimeric immunoglobulins are the product of fused genes derived from different species; a "humanized" chimera typically has framework regions (FRs) from a human immunoglobulin and one or more CDRs from a non-human source.

[0256] In certain aspects, portions of the heavy and / or light chains are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods of chimeric antibodies, see, for example, U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), each of which is incorporated herein by reference in its entirety. CDR importing is described, for example, in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated by reference for all purposes.

[0257] In some embodiments, minimizing the antibody polypeptide sequences from non-human species can optimize chimeric antibody function and reduce immunogenicity. Specific amino acid residues from non-antigen recognition regions of non-human antibodies are modified to be homologous to corresponding residues in human antibodies or isotypes. An example is a "CDR transplant" antibody, in which the antibody comprises one or more CDRs from a specific species or belonging to a specific antibody class or subclass, and the rest of the antibody chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass. For use in humans, the variable region consisting of CDR1, CDR2 and part of CDR3 of the light chain and heavy chain variable regions of non-human immunoglobulins is introduced into the human antibody framework region, replacing the natural antigen receptor of human antibodies with non-human CDRs. In some cases, the corresponding non-human residues replace the framework region residues of human immunoglobulins. In addition, humanized antibodies may contain residues not found in the receptor antibody or donor antibody to further improve performance. Humanized antibodies may also contain at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. See, for example, Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeye et al., Science 239: 1534-36 (1988).

[0258] In vivo antibodies are intracellularly localized immunoglobulins that bind to intracellular antigens, in contrast to secreted antibodies, which bind antigens in the extracellular space.

[0259] Polyclonal antibody preparations usually include different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host (such as a rabbit or goat) is immunized with an antigen or antigen fragment, usually with an adjuvant and, if necessary, coupled to a carrier. Antibodies to the antigen are then collected from the host's serum. Polyclonal antibodies can be affinity purified against the antigen to make them monospecific.

[0260] A monoclonal antibody or "mAb" refers to an antibody obtained from a homogeneous population of antibodies from exclusive parental cells, i.e., the population is identical except for naturally occurring mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.

[0261]

C. Functional antibody fragments and antigen-binding fragments

[0262] 【1. Antigen-binding fragment】

[0263] Certain aspects relate to antibody fragments, such as antibody fragments that bind to a TdT peptide. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to an antigen. These fragments consist of various arrangements of variable region heavy chains (VH) and / or light chains (VL); in some embodiments, constant region heavy chain 1 (CH1) and light chain (CL). In some embodiments, they lack the Fc region consisting of heavy chain 2 (CH2) and 3 (CH3) domains. Embodiments of antigen-binding fragments and modifications thereof may include: (i) Fab fragment types consisting of VL, VH, CL and CH1 domains; (ii) Fd fragment types consisting of VH and CH1 domains; (iii) Fv fragment types consisting of VH and VL domains; (iv) single domain fragment type dAb consisting of a single VH or VL domain (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003); (v) isolated complementarity determining region (CDR) regions. These terms are described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22: 189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and in Day, ED, Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, NY (1990); Antibodies, 4: 259-277 (2015), each of which is incorporated by reference in its entirety.

[0264] Antigen binding fragments also include fragments of antibodies that retain exactly, at least or at most, 1, 2 or 3 complementary determining regions (CDRs) from the light chain variable region. Fusion of CDR-containing sequences to Fc regions (or their CH2 or CH3 regions) is included within the scope of this definition, including, for example, scFvs fused directly or indirectly to Fc regions are also included within the scope of this definition.

[0265] The term Fab fragment (also referred to as "Fab") refers to a monovalent antigen-binding fragment of an antibody comprising the VL, VH, CL and CH1 domains. The term Fab' fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than the Fab fragment. For example, a Fab' fragment includes the VL, VH, CL and CH1 domains and all or part of the hinge region. The term F(ab')2 fragment refers to a divalent antigen-binding fragment of a monoclonal antibody, consisting of two Fab' fragments, which are connected by a disulfide bond at the hinge region. For example, a F(ab')2 fragment includes all or part of two VH and VL domains, and may further include all or part of two CL and CH1 domains.

[0266] The term Fd fragment refers to a heavy chain fragment of a monoclonal antibody, which comprises all or part of VH, including CDRs. The Fd fragment may further include a CH1 region sequence.

[0267] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody, including all or part of VL and VH, and without CL and CH1 domains. VL and VH include CDRs, etc. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions are connected by a flexible linker to form a single polypeptide chain to form an antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference as a whole. The term (scFv) 2 refers to a bivalent or bispecific sFv polypeptide chain, the C-terminus of which contains an oligomerization domain, which is separated from the sFv by a hinge region (Pack et al., 1992). The oligomerization domain includes a self-associated α-helix, such as a leucine zipper, which can be further stabilized by additional disulfide bonds. (scFv) 2 fragments are also referred to as "miniantibodies" or "miniantibodies".

[0268] Single domain antibodies are antigen binding fragments that contain only a VH or VL domain. In some cases, two or more VH regions are covalently linked with a peptide linker to produce a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0269] 【2. Fragment antigen binding region, Fab】

[0270] Fab polypeptides disclosed herein include Fab antigen-binding fragments of antibodies. Unless otherwise specifically stated, the term "Fab" refers to a polypeptide that does not include the Fc portion of an antibody. Fab can be coupled to a polypeptide comprising other components, such as further antigen-binding domains, costimulatory domains, linkers, peptide spacers, transmembrane domains, endodomains, and accessory proteins. Fab polypeptides can be generated using conventional techniques known in the art and are well described in the literature.

[0271] 【3. Fragment crystallizable region, Fc】

[0272] The Fc region comprises two heavy chain fragments, which comprise the CH2 and CH3 domains of an antibody. The two heavy chain fragments are bound together by two or more disulfide bonds and the hydrophobic interactions of the CH3 domains. The term "Fc polypeptide" as used herein includes native and mutant protein forms of polypeptides derived from the Fc region of an antibody. A truncated form of such polypeptide comprising a hinge region that promotes dimerization is included.

[0273] [D. Polypeptide having antibody CDR and scaffold domain displaying CDR]

[0274] Antigen binding peptide scaffolds, such as complementary determining regions (CDRs), are used to generate protein binding molecules according to the embodiments. Generally, one skilled in the art can determine the type of protein scaffold on which at least one CDR is introduced. It is well known that the scaffold preferably must meet many criteria, such as: good phylogenetic conservation; known three-dimensional structure; small size; little or no post-transcriptional modification; and / or easy production, expression and purification (Skerra, J Mol Recognit, 13: 167-87 (2000)).

[0275] The protein scaffold may be derived from, but is not limited to, fibronectin type III FN3 domain (referred to as "monomer"), fibronectin type III domain 10, lipocalin, anti-calcitonin, the Z-domain of Staphylococcus aureus protein A, thioredoxin A, or a protein with a repeating motif, such as "ankyrin repeats," "armadillo repeats," "leucine-rich repeats," and "tetratricopeptide repeats." Such proteins are described in U.S. Patent Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. WO2006 / 056464, each of which is specifically incorporated herein by reference in its entirety. Scaffolds derived from toxins of scorpions, insects, plants, mollusks, etc., as well as protein inhibitors of neuronal NO synthase (PIN) may also be used.

[0276]

E. Antibody Binding

[0277] The term "selective binding agent" refers to a molecule that binds to an antigen. Non-limiting examples include antibodies, antigen-binding fragments, scFv, Fab, Fab', F(ab')2, single-chain antibodies, peptides, peptide fragments, and proteins.

[0278] The term "binding" refers to direct binding between two molecules, for example due to covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. "Immunoreactivity" means that a selective binding agent or antibody of interest will bind to an antigen present in a biological sample. The term "immune complex" refers to the combination formed when an antibody or selective binding agent binds to an epitope on an antigen.

[0279] 【1. Affinity / Affinity】

[0280] The term "affinity" refers to the strength with which an antibody or selective binding agent binds to an epitope. In an antibody binding reaction, this is expressed as the affinity constant (Ka or ka is sometimes referred to as the association constant) of any given antibody or selective binding agent. Affinity is measured as a comparison of the binding strength of an antibody to its associated antigen with the binding strength of the antibody to an unrelated amino acid sequence. Affinity can be expressed, for example, as a 20-fold higher binding capacity of an antibody to its antigen than to an unrelated amino acid sequence. The term "affinity" as used herein refers to the resistance of a complex of two or more reagents to dissociation after dilution. The terms "immunoreactivity" and "preferential binding" are used interchangeably herein with respect to antibodies and / or selective binding agents.

[0281] There are several experimental methods that can be used by those skilled in the art to assess the binding affinity of any given antibody or selective binding agent for its antigen. This is usually accomplished by measuring the equilibrium dissociation constant (KD or Kd), using the equation KD = k off / k on =[A][B] / [AB]. Term k off The k is the rate of dissociation between the antibody and the antigen per unit time and is related to the concentration of the antibody and antigen present in the solution in unbound form at equilibrium. on It is the rate at which antibody and antigen bind per unit time and is related to the concentration of bound antigen-antibody complexes at equilibrium. The unit used to measure KD is mol / L (molar concentration or M) or concentration. The Ka of an antibody is the inverse of the KD and is determined by the equation Ka = 1 / KD. Some examples of experimental methods that can be used to determine KD values ​​include: enzyme-linked immunosorbent assay (ELISA), isothermal titration calorimetry (ITC), fluorescence anisotropy, surface plasmon resonance (SPR), and affinity capillary electrophoresis (ACE). The affinity constant (Ka) of an antibody is the inverse of the KD and is determined by the equation Ka = 1 / KD.

[0282] Antibodies contemplated as useful in certain embodiments may have a 6 , 10 7 , 10 8 , 10 9 or 10 10 M or any range derivable therein. Similarly, in some embodiments, an antibody may have an affinity constant (Ka) of about, at least, or at most about 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or any range of dissociation constants derivable therein. These values ​​are reported for the antibodies discussed herein, and the same assays can be used to assess the binding properties of such antibodies. When the dissociation constant (KD) is ≤10 -8 M, an antibody of the invention is said to "specifically bind" to its target antigen. -9 M, the antibody specifically binds to the antigen with "high affinity", and when KD is ≤5×10 -10 M, the antibody specifically binds the antigen with "very high affinity".

[0283] 【2. Epitope specificity】

[0284] The epitope of an antigen is a specific region of an antigen to which an antibody has binding affinity. In the case of a protein or polypeptide antigen, an epitope is a specific residue (or specific amino acid or protein fragment) to which an antibody binds with high affinity. An antibody does not necessarily contact every residue in a protein. Every amino acid substitution or deletion in a protein does not necessarily affect binding affinity. For the purposes of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen that a B cell and / or T cell reacts or recognizes. A polypeptide epitope can be formed by continuous and non-continuous amino acids and is juxtaposed to the tertiary fold of a polypeptide. An epitope typically includes at least 3 amino acids, typically 5 to 10 amino acids, and has a unique spatial conformation.

[0285] The epitope specificity of an antibody can be determined in a variety of ways. For example, one method involves testing a set of overlapping peptides that are 15 amino acids across the entire sequence of the protein and differ in increments of a small number of amino acids (e.g., 3 to 30 amino acids). The peptides are fixed in separate wells of a microtiter dish. For example, fixation can be accomplished by biotinylating one end of the peptide. This process may affect the affinity of the antibody for the epitope, so different samples of the same peptide can be biotinylated at the N and C termini and fixed in separate wells for comparison. This is useful for identifying end-specific antibodies. Alternatively, other peptides that terminate at specific target amino acids may be included. This method can be used to identify end-specific antibodies against internal fragments. Screen antibodies or antigen-binding fragments to bind to each of the various peptides. An epitope is defined as an amino acid fragment common to all peptides to which an antibody shows high affinity binding.

[0286] 【3. Modification of Antibody Antigen Binding Domain】

[0287] It is understood that the antibodies of the present disclosure can be modified so that they are substantially identical to the antibody polypeptide sequences or fragments thereof and still bind to the epitopes of the present invention. When optimally aligned using programs such as Clustal Omega, IGBLAST, GAP or BESTFIT with default gap weights, polypeptide sequences are "substantially identical" if they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, or any range thereof.

[0288] As discussed herein, minor changes in the amino acid sequence of an antibody or antigen-binding region thereof are contemplated as being encompassed by the present invention, provided that the changes in the amino acid sequence maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and most preferably at least 99% sequence identity. In particular, conservative amino acid substitutions are contemplated.

[0289] Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are often divided into families based on the chemical properties of their side chains; for example, acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). For example, it is reasonable to expect that isolated substitutions of a leucine moiety with an isoleucine or valine moiety, or similar substitutions with structurally related amino acids in the same family, will not have a significant effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve amino acids within a framework site. Whether an amino acid change results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Standard ELISA, surface plasmon resonance (SPR) or other antibody binding assays can be performed by one skilled in the art to quantitatively compare antigen binding affinity between an unmodified antibody and any polypeptide derivative that is conservatively substituted by any of several methods available to one skilled in the art.

[0290] Fragments or analogs of antibodies or immunoglobulin molecules can be easily prepared by those skilled in the art. Preferred amino and carboxyl termini of fragments or analogs appear near the boundaries of functional domains. By comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases, structural and functional domains can be identified. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that appear in other proteins of known structure and / or function. Standard methods for identifying protein sequences that fold into known three-dimensional structures are available to those skilled in the art; Dill and McCallum., Science 338: 1042-1046 (2012). Several algorithms for predicting protein structures and gene sequences encoding these structures have been developed, many of which can be found in the National Center for Biotechnology Information (at www.ncbi.nlm.nih.gov / guide / proteins / ) and the Bioinformatics Resource Portal (at www.expasy.org / proteomics / ). Thus, the above examples demonstrate that one skilled in the art can recognize sequence motifs and structural conformations that can be used to define structural and functional domains in accordance with the present invention.

[0291] Framework modifications may be made to antibodies to reduce immunogenicity, for example, by "backmutating" one or more framework residues to the corresponding germline sequence.

[0292] It is also contemplated that antigen binding domains can be multispecific or multivalent by multimerizing antigen binding domains having pairs of VH and VL regions that bind the same antigen (multivalent) or different antigens (multispecific).

[0293]

F. Chemical modification of antibodies

[0294] In certain aspects, glycosylation variants of antibodies are also contemplated, in which the number and / or type of glycosylation sites have been altered relative to the amino acid sequence of the parent polypeptide. The glycosylation of a polypeptide can be altered, for example, by modifying one or more glycosylation sites within the polypeptide sequence to increase the affinity of the polypeptide for an antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861). In certain embodiments, an antibody protein variant comprises a greater or lesser number of N-linked glycosylation sites than a native antibody. The N-linked glycosylation site is characterized by the following sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. Substitution of amino acid residues produces this sequence, providing a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent the addition of an N-linked carbohydrate chain present in the native polypeptide. For example, glycosylation can be reduced by deleting asparagine or replacing asparagine with a different amino acid. In other embodiments, one or more new N-linked glycosylation sites are produced. Antibodies typically have an N-linked glycosylation site in the Fc region.

[0295] Other antibody variants include cysteine ​​variants, in which one or more cysteine ​​residues of the parent or native amino acid sequence are deleted from or substituted for another amino acid (e.g., serine). Cysteine ​​variants are particularly useful when the antibody must be refolded into a conformation with biological activity. Cysteine ​​variants may have fewer cysteine ​​residues than native antibodies and generally have an even number to minimize interactions caused by unpaired cysteines.

[0296] In certain aspects, the polypeptide can be pegylated to increase biological half-life by reacting the polypeptide with PEG or a reactive ester or aldehyde derivative of PEG under conditions where one or more polyethylene glycol (PEG) groups are attached to the polypeptide. Pegylated polypeptides can be achieved by acylation or alkylation with reactive PEG molecules (or similar reactive water-soluble polymers). Methods for pegylation of proteins are known in the art and can be applied to the polypeptides of the present invention to obtain pegylated derivatives of antibodies. For example, see EP 0 154 316 and EP 0 401 384. In certain aspects, the antibody is coupled or otherwise linked to transthyretin (TTR) or a TTR variant. TTR or a TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols, and polyvinyl alcohol. As used herein, the term "polyethylene glycol" is intended to include any of the forms of PEG that have been used to derivatize other proteins.

[0297] 【1.Join】

[0298] Also provided herein are derivatives of the antibodies and antigen-binding fragments described herein. A derivatized antibody or fragment thereof may comprise any molecule or substance that imparts the desired properties to the antibody or fragment. A derivatized antibody may include, for example, a detectable (or labeling) portion (e.g., a radioactive, colorimetric, antigenic or enzyme molecule, or a detectable microbead), a molecule that binds to another molecule (e.g., biotin or streptavidin), a therapeutic or diagnostic portion (e.g., a radioactive, cytotoxic or pharmaceutically active portion), or a molecule that increases the suitability of the antibody for a particular use (e.g., administration to a subject (e.g., a human subject) or other in vivo or in vitro use).

[0299] Alternatively, the antibody or the immune portion of the antibody can be chemically coupled or expressed as a fusion protein with other proteins. In some aspects, the polypeptide can be chemically modified by coupling or fusion with serum proteins (such as human serum albumin) to increase the half-life of the resulting molecule. For example, see EP 0322094 and EP 0 486 525. In some aspects, the polypeptide can be coupled with a diagnostic agent and used for diagnosis, for example, to monitor the development or progress of the disease and determine the efficacy of a given treatment regimen. In some aspects, the polypeptide can also be coupled with a therapeutic agent to provide a treatment combined with the therapeutic effect of the polypeptide. Other suitable coupling molecules include ribonucleases (RNases), DNase I, antisense nucleic acids, inhibitory RNA molecules (such as siRNA molecules), immunostimulatory nucleic acids, aptamers, ribozymes, triplex formation molecules and external guide sequences. Functional nucleic acid molecules can serve as effectors, inhibitors, regulators and stimulants of the specific activity possessed by the target molecule, or functional nucleic acid molecules can have de novo synthesis activity independent of any other molecule.

[0300] In some aspects, disclosed are antibodies and antibody-like molecules that are connected to at least one reagent to form an antibody conjugate or payload. In order to improve the efficacy of the antibody molecule as a diagnostic or therapeutic agent, at least one desired molecule or part is usually connected or covalently bound or coordinated. Such a molecule or part can be, but is not limited to, at least one effector or reporter molecule. Effector molecules include molecules with desired activity, such as cytotoxic activity. Non-limiting examples of effector molecules include toxins, therapeutic enzymes, antibiotics, radiolabeled nucleotides, etc. In contrast, reporter molecules are defined as any part that can be detected using an assay. Non-limiting examples of reporter molecules coupled to antibodies include enzymes, radiolabels, haptens, fluorescent markers, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles, or ligands.

[0301] 【2.Joining type】

[0302] Some examples of antibody conjugates are those conjugates in which antibodies are connected to detectable labels. "Detectable label" refers to compounds and / or elements that can be detected due to their specific functional properties and / or chemical properties, using which antibodies can be detected, and / or further quantified as needed. Examples of detectable labels include, but are not limited to, radioisotopes, fluorescent agents, semiconductor nanocrystals, chemiluminescent agents, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, metal sols, ligands (e.g., biotin, streptavidin or haptens), etc. Specific examples of labels include, but are not limited to, horseradish peroxidase (HRP), fluorescein, fluorescein isothiocyanate, rhodamine, dansyl chloride, hydroxycoumarin, dimethyl acridinium ester (DMAE), Texas Red, luminol, NADPH and α or β-galactosidase. Antibody conjugates include antibody conjugates primarily used in vitro, in which antibodies are connected to secondary binding ligands and / or enzymes to produce colored products when in contact with chromogenic substrates. Examples of suitable enzymes include, but are not limited to, urease, alkaline phosphatase, (horseradish) peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and / or avidin and streptavidin compounds. The use of such tags is well known to those skilled in the art and is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each of which is incorporated herein by reference in its entirety. Molecules containing azide groups can also be used to form covalent bonds with proteins by active nitrase intermediates generated by low-intensity ultraviolet light (Potter & Haley, 1983).

[0303] In some aspects, the immunoconjugate envisioned comprises an antibody or its antigen-binding fragment and a cytotoxic agent such as a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatic toxin or a fragment thereof of a bacterial, fungal, plant or animal origin) or a radioactive isotope (i.e., a radioactive conjugate). In this way, the target agent can directly target cells carrying cell surface antigens. The antibody and the agent can be bound by non-covalent interactions (e.g., electrostatic forces) or covalent bonds. Various joints known in the art can be used to form an immunoconjugate. In addition, the immunoconjugate can be provided in the form of a fusion protein. On the one hand, the antibody can be coupled to various therapeutic substances to target cell surface antigens. Examples of coupling agents include, but are not limited to, metal chelate complexes, drugs, toxins and other effector molecules, such as cytokines, lymphokines, chemokines, immunomodulators, radiosensitizers, asparaginase, carboborin and radioactive halogens.

[0304] In an antibody drug conjugate (ADC), an antibody (Ab) is coupled to one or more drug moieties (D) via a linker (L). ADCs can be prepared by a variety of routes, using organic chemical reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting a nucleophilic group of the antibody with a divalent linker reagent to form Ab-L via a covalent bond, which is then reacted with the drug moiety D; (2) reacting a nucleophilic group of the drug moiety with a divalent linker reagent to form DL via a covalent bond, which is then reacted with a nucleophilic group of the antibody. Antibody drug conjugates can also be produced by modifying an antibody to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug. Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be prepared, for example, by recombinant techniques or peptide synthesis. The length of DNA can include regions encoding the two portions of the conjugate, which are adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate. In another aspect, the antibody can be coupled to a "receptor" (e.g., streptavidin) for tumor or cancer cell pretargeting, where the antibody-receptor conjugate is administered to the patient, unbound conjugate is then removed from the circulation using a clearing agent, and then a "ligand" (e.g., avidin) coupled to a cytotoxic agent (e.g., radionucleotide) is administered.

[0305] Examples of antibody-drug conjugates known to those skilled in the art are pro-drugs that can be used for local delivery of cytotoxic or cytostatic drugs, i.e., drugs that kill or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Res. 19:605-614 (1999); Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26:151-172 (1997); U.S. Pat. No. 4,975,278). In contrast, systemic administration of these unconjugated drugs may produce unacceptable levels of toxicity to normal cells and target tumor cells (Baldwin et al., Lancet 1: 603-5 (1986); Thorpe, (1985) "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," In: Monoclonal Antibodies '84: Biological and Clinical Applications, A. Pincera et al., (eds.) pp. 475-506). It has been reported that both polyclonal and monoclonal antibodies are useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21: 183-87 (1986)).

[0306] In certain aspects, ADCs include covalent or aggregated conjugates of antibodies or antigen-binding fragments thereof with other proteins or polypeptides, such as by expressing a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of an antibody polypeptide. For example, the conjugated peptide can be a heterologous signal (or leader) polypeptide, such as a yeast alpha factor leader, or a peptide, such as an epitope tag (e.g., V5-His). Fusion proteins containing antibodies may include peptides (e.g., poly-His) added to facilitate antibody purification or identification. Antibody polypeptides can also be conjugated to (Sigma-Aldrich, St. Louis, Mo.) peptide linkage, as described in Hopp et al., Bio / Technology 6: 1204 (1988) and U.S. Pat. No. 5,011,912. Oligomers containing one or more antibody polypeptides can be used as antagonists. The oligomers can be in the form of dimers, trimers, or higher oligomers that are covalently or non-covalently linked. It is contemplated that oligomers containing two or more antibody polypeptides can be used. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc. In certain aspects, the oligomer contains multiple antibody polypeptides that are linked by covalent or non-covalent interactions between peptide portions fused to the antibody polypeptides. Such peptides can be peptide linkers (spacers), or peptides that have oligomerization-promoting properties. Leucine zippers and certain antibody-derived polypeptides are peptides that can promote oligomerization of antibody polypeptides attached thereto, as described below.

[0307] 【3.Joining method】

[0308] Several methods known in the art are used to connect or couple an antibody to its conjugate moiety. Some attachment methods involve the use of metal chelates, for example, using organic chelators such as diethylenetriaminepentaacetic anhydride (DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and / or tetrachloro-3-6-diphenylglycan-3-yl (U.S. Pat. Nos. 4,472,509 and 4,938,948, both incorporated herein by reference). Monoclonal antibodies can also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates can also be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothioalkanes (IT), bifunctional derivatives of iminoesters (such as hexanedimethyl hydrochloride), active esters (such as disuccinimide), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazo derivatives (such as BOS (p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In certain aspects, it is contemplated to derivatize immunoglobulins by selectively introducing sulfhydryl groups into the Fc region of the immunoglobulin, using reaction conditions that do not alter the antibody combining site. Antibody conjugates produced according to the method are disclosed to have higher longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is coupled to carbohydrate residues of the Fc region, has been described in the literature (O'Shannessy et al., 1987).

[0309]

III. Antibody Production

[0310] A. Antibody production

[0311] Methods for preparing and characterizing antibodies for use in diagnostic and detection assays, purification, and as therapeutic agents are well known in the art, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745 (e.g., see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference). These antibodies can be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, single domain antibodies, dimer or trimer antibody fragment constructs, miniantibodies, or functional fragments that bind to relevant antigens. In certain aspects, polypeptides, peptides, proteins, and immunogenic fragments thereof used in various embodiments can also be synthesized in solution or on a solid support according to conventional techniques. See, for example, Stewart and Young, (1984); Tarn et al, (1983); Merrifield, (1986); and Barany and Merrifield (1979), each of which is incorporated herein by reference in its entirety.

[0312] In short, polyclonal antibodies are prepared by immunizing an animal with an antigen or a portion thereof and collecting antiserum from the immunized animal. Antigens may be altered compared to antigen sequences found in nature. In some embodiments, variant or altered antigen peptides or polypeptides are used to produce antibodies. Inoculum is usually prepared by dispersing an antigen composition in a physiologically tolerable diluent to form an aqueous composition. Antiserum is subsequently collected by methods known in the art, and the serum can be used as is for various applications, or the antibody components required for purification by recognized methods, such as affinity chromatography (Harlow and Lane, Antibodies: A Laboratory Manual 1988).

[0313] Methods for preparing monoclonal antibodies are also well known in the art (Kohler and Milstein, 1975; Harlow and Lane, 1988, U.S. Pat. No. 4,196,265, all of which are incorporated herein by reference for all purposes). In general, the technique involves immunizing a suitable animal with a selected immunogenic component (e.g., a purified or partially purified protein, polypeptide, peptide or domain). The antibody-producing B cells or all dissociated spleen cells produced by the immunized animal are then induced to fuse with cells of an immortalized cell line to form a hybridoma. Myeloma cell lines suitable for fusion procedures for producing hybridomas are preferably non-antibody-producing and have high fusion efficiency and enzyme defects, which prevent them from growing in certain selective culture media (hybridomas) that only support the growth of the desired fused cells. In general, the fusion partner includes a property that allows the resulting hybridoma to be selected using a specific culture medium. For example, the fusion partner may be sensitive to hypoxanthine / aminopterin / thymidine (HAT). The method of producing antibody-producing spleen or lymph node cells and myeloma cell hybrids generally includes mixing somatic cells with myeloma cells in the presence of an agent (chemical or electrical) that promotes cell membrane fusion. Next, hybridomas can be selected by culturing cells by monoclonal dilution in microtiter plates and then testing individual clone supernatants (after two to three weeks) to obtain the desired reactivity. Fusion procedures, immunization protocols, and separation of immune spleen cells for fusion techniques for preparing hybridomas are known in the art.

[0314] Other techniques for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes, the use of molecular cloning methods to generate nucleic acids or polypeptides, the selected lymphocyte antibody method (SLAM) (see, e.g., Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996), preparing combinatorial immunoglobulin phagemid libraries from RNA isolated from the spleen of an immunized animal and selecting phagemids that express the appropriate antibody, or using Cre-mediated site-specific recombination to generate cells expressing antibodies from cellular genomic sequences containing modified immunoglobulin sites (see, e.g., US 6,091,001).

[0315] The monoclonal antibodies can be further purified using filtration, centrifugation, and various chromatographic methods (such as HPLC or affinity chromatography). The monoclonal antibodies can be further screened or optimized for properties related to specificity, affinity, half-life, immunogenicity, binding association, binding dissociation, or overall functional properties relative to infection treatment. Thus, the monoclonal antibodies may have altered amino acid sequences of CDRs, including insertions, deletions, or substitutions for conservative or non-conservative amino acids.

[0316] The immunogenicity of a particular immunogenic composition can be enhanced by using a non-specific stimulant called an adjuvant. Adjuvants that can be used according to the embodiments include, but are not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). Exemplary adjuvants may include complete Freund's adjuvant (a non-specific stimulator of an immune response containing inactivated Mycobacterium tuberculosis), incomplete Freund's adjuvant, and / or aluminum hydroxide adjuvant. In addition to adjuvants, it may be necessary to use a biological response modifier (BRM) in combination, such as, but not limited to, cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ), cytokines (such as beta-interferon, IL-2 or IL-12), or genes encoding proteins involved in immune auxiliary functions, such as B-7. Phage display systems can be used to amplify antibody molecule populations in vitro. Saiki, et al., Nature 324:163 (1986); Scharf et al., Science 233:1076 (1986); U.S. Patent Nos. 4,683,195 and 4,683,202; Yang et al., J Mol Biol. 254:392 (1995); Barbas, III et al., Methods: Comp. Meth Enzymol. (1995) 8:94; Barbas, III et al., Proc Natl Acad Sci USA 88:7978 (1991).

[0317]

B. Fully human antibody production

[0318] Methods for preparing fully human antibodies. The use of fully human antibodies can minimize the immunogenicity and allergic reactions that may be caused by the administration of non-human monoclonal antibodies as therapeutic agents. In one embodiment, human antibodies can be produced in non-human transgenic animals, for example, transgenic mice that can produce multiple human protein antibody isotypes (e.g., IgG, IgA and / or IgE) by undergoing VDJ recombination and isotype conversion. Therefore, this aspect is applicable to antibodies, antibody fragments and pharmaceutical compositions thereof, and is also applicable to non-human transgenic animals, B cells, host cells and hybridomas that produce monoclonal antibodies. The application of human antibodies includes, but is not limited to, in vivo or in vitro detection of cells expressing the expected protein, pharmaceutical preparations containing antibodies of the present invention, and methods for treating diseases by administering antibodies.

[0319] Fully human antibodies can be produced by immunizing transgenic animals (usually mice), which are able to produce a range of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose generally have 6 or more consecutive amino acids and can be selected to be coupled to a carrier (such as a hapten). For example, see Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-2555 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggermann et al., Yearin Immunol. 7: 33 (1993). In one example, transgenic animals are produced by disabling the endogenous mouse immunoglobulin loci encoding mouse immunoglobulin heavy and light chains, and inserting large fragments of human genomic DNA containing loci encoding human heavy and light chain proteins into the mouse genome. Then hybridize partially modified animals with less than complete human immunoglobulin loci to obtain animals with all desired immune system modifications. When an immunogen is administered, these transgenic animals produce antibodies that are immunospecific for the immunogen, but have human rather than murine amino acid sequences, including variable regions. For more details on such methods, see, for example, International Patent Application Publication Nos. WO 96 / 33735 and WO 94 / 02602, which are hereby incorporated by reference in their entirety. U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; describe other methods for making human antibodies related to transgenic mice; International Patent Application Publication Nos. WO 91 / 10741 and WO 90 / 04036; and European Patent Nos. EP 546073B1 and EP546073A1, all of which are hereby incorporated by reference in their entirety.

[0320] The transgenic mice described above, referred to herein as "HuMAb" mice, contain human immunoglobulin gene minisites encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate endogenous μ and κ chain sites (Lonberg et al., Nature 368: 856-859 (1994)). Thus, the mice exhibit reduced expression of mouse IgM or κ chains, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg et al., supra; Lonberg and Huszar, Intern. Ref. Immunol. 13: 65-93 (1995); Harding and Lonberg, Ann. NY Acad. Sci. 764: 536-546 (1995)). HuMAb mouse preparation is described in Taylor et al., Nucl. Acids Res. 20:6287-6295 (1992); Chen et al., Int. Immunol. 5:647-656 (1993); Tuaillon et al., J. Immunol. 152:2912-2920 (1994); Lonberg et al., supra; Lonberg, Handbook of Exp. Pharmacol. 113:49-101 (1994); Taylor et al., Int. Immunol. 6:579-591 (1994); Lonberg and Huszar, Intern. Ref. Immunol. 13:65-93 (1995); Harding and Lonberg, Ann. NY Acad. Sci. 764:536-546 (1995); Fishwild et al., Nat. Biotechnol. 14:845-851 (1996); are described in detail. The above references are incorporated herein by reference in their entirety for all purposes. See further U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; 5,770,429; and 5,545,807; and International Patent Application Publication Nos. WO 93 / 1227; WO 92 / 22646; and WO 92 / 03918, all of which disclosures are hereby incorporated by reference in their entirety for all purposes.Techniques for producing human antibodies in these transgenic mice are also disclosed in WO 98 / 24893 and Mendez et al., Nat. Genetics 15:146-156 (1997), which are incorporated herein by reference. For example, HCo7 and HCo12 transgenic mouse strains can be used to generate human antibodies.

[0321] Using hybridoma technology, antigen-specific humanized monoclonal antibodies with desired specificity can be produced and selected from transgenic mice such as those described above. Such antibodies can be cloned and expressed using appropriate vectors and host cells, or they can be harvested from cultured hybridoma cells. Fully human antibodies can also be derived from phage display libraries (such as Hoogenboom et al., J. Mol. Biol. 227: 381 (1991); and Marks et al., J. Mol. Biol. 222: 581 (1991)). International Patent Application Publication No. WO 99 / 10494 (incorporated herein by reference) describes such a technique, which uses this method to separate high affinity and functional agonistic antibodies for MPL and msk receptors.

[0322]

C. Antibody fragment production

[0323] Antibody fragments that retain the ability to recognize the target antigen will also find use here. Many antibody fragments known in the art contain antigen binding sites, are able to exhibit the immunological binding properties of intact antibody molecules, and can subsequently be modified by methods known in the art. Functional fragments, including only the variable regions of the heavy and light chains, can also be produced using standard techniques, such as preferential proteolytic cleavage of immunoglobulin molecules or recombinant production. These fragments are referred to as Fv. For example, see Inbar et al., Proc. Nat. Acad. Sci. USA 69: 2659-2662 (1972); Hochman et al., Biochem. 15: 2706-2710 (1976); and Ehrlich et al., Biochem. 19: 4091-4096 (1980).

[0324] Single-chain variable fragments (scFvs) can be prepared by fusing DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). scFvs can form antigen-binding monomers or multimers (e.g., dimers, trimers, or tetramers), depending on the length of the flexible linker between the two variable domains (Kort et al., Prot. Eng. 10: 423 (1997); Kort et al., Biomol. Eng. 18: 95-108 (2001)). By combining different polypeptides comprising VL and VH, multimeric scFvs that bind to different epitopes can be formed (Kriangkum et al., Biomol. Eng. 18: 31-40 (2001)). Antigen-binding fragments are typically produced by recombinant DNA methods known to those skilled in the art. Although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be linked together using recombinant methods through synthetic linkers, thereby enabling them to be made into a single-chain polypeptide (referred to as single-chain Fv (sFv or scFv); see, for example, Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Design criteria include determining the appropriate length of the distance between the C-terminus of one chain and the N-terminus of the other chain, wherein the linker is generally formed by small hydrophilic amino acid residues that do not tend to coil or form secondary structures. Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include inserted glutamic acid and lysine residues to improve solubility. The practical screening of antigen-binding fragments is the same as that of intact antibodies. Such fragments include fragments obtained by amino-terminal and / or carboxyl-terminal deletions, wherein the remaining amino acid sequence is substantially the same as the corresponding positions derived in the naturally occurring sequence, for example, inferred from the full-length cDNA sequence.

[0325] Antibodies can also be generated using peptide analogs of the epitope determinants disclosed herein, which can be composed of non-peptide compounds having properties similar to the template peptide. These types of non-peptide compounds are referred to as "peptide mimetics" or "mimetic peptides". Fauchere, J. Adv. Drug Res. 15: 29 (1986); Veber and Freidinger TINS ​​p. 392 (1985); and Evans et al., J. Med. Chem. 30: 1229 (1987). Liu et al. (2003) also described "antibody-like binding peptide mimetics" (ABiPs), which are peptides that act as streamlined antibodies, with longer serum half-lives and certain advantages of less cumbersome synthetic methods. These analogs can be peptides, non-peptides, or a combination of peptide and non-peptide regions. Fauchere, Adv. Drug Res. 15: 29 (1986); Veber and Freidiner, TINS ​​p. 392 (1985); and Evans et al., J. Med. Chem. 30: 1229 (1987), which are incorporated herein by reference in their entirety for any purpose. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce similar therapeutic or preventive effects. Such compounds are usually developed with the help of computerized molecular modeling. Generally, the peptidomimetic compounds of the present invention are proteins that are structurally similar to antibodies, exhibiting the desired biological activity, such as the ability to bind to proteins, but having one or more peptide bonds that are optionally substituted with bonds selected from the following options by methods well known in the art: -CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2- and -CH2SO-. In certain embodiments of the invention, one or more amino acids of a consensus sequence can be systematically substituted with the same type of D-amino acid (e.g., D-lysine substituted for L-lysine) to produce a more stable protein. In addition, constrained peptides comprising a consensus sequence or substantially identical consensus sequence variations can be produced by methods known in the art (Rizo and Gierasch, Ann. Rev. Biochem. 61: 387 (1992), incorporated herein by reference), for example, by adding a cysteine ​​residue capable of forming an intramolecular disulfide bond to cyclize the peptide.

[0326] Once generated, the phage display library can be used to improve the immunological binding affinity of the Fab molecule using known techniques. For example, see Figini et al., J. Mol. Biol. 239: 68 (1994). The coding sequences of the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used.

[0327] [IV. Obtaining Encoded Antibodies]

[0328] In certain aspects, there are nucleic acid molecules encoding TdT peptide antibody polypeptides (e.g., heavy or light chain, variable domain only or full length). These can be produced by methods known in the art, for example, isolated from B cells of immunized and isolated mice, displayed on phage, expressed in any suitable recombinant expression system and allowed to assemble to form antibody molecules.

[0329] A. Expression

[0330] Nucleic acid molecules can be used to express large amounts of recombinant antibodies or to produce chimeric antibodies, single-chain antibodies, immunoadhesins, diabodies, mutant antibodies, and other antibody derivatives. If the nucleic acid molecule is derived from a non-human, non-transgenic animal, the nucleic acid molecule can be used for antibody humanization.

[0331] [1. Carrier]

[0332] In some aspects, the contemplated expression vectors contain nucleic acid molecules encoding polypeptides of the desired sequence or portions thereof (e.g., fragments comprising one or more CDRs or one or more variable region domains). Expression vectors comprising nucleic acid molecules may encode heavy chains, light chains, or antigen binding portions thereof. In some aspects, expression vectors comprising nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences for controlling transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0333] In order to express antibodies or antigen-binding fragments thereof, DNA encoding partial or full-length light and heavy chains is inserted into an expression vector so that the gene region is operably linked to transcription and translation control sequences. In some aspects, the vector encoding a functionally complete human CH or CL immunoglobulin sequence has appropriate restriction sites, so any VH or VL sequence can be easily inserted and expressed. Generally, the expression vector used in any host cell contains sequences for plasmid or virus maintenance and cloning and expression of exogenous nucleotide sequences. Such sequences are collectively referred to as "flanking sequences" and generally include one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a polypeptide secretion leader sequence, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Such sequences and methods of use thereof are well known in the art.

[0334] 【2. Expression system】

[0335] There are many expression systems, which at least comprise some or all of the expression vectors discussed above. Systems based on prokaryotes and / or eukaryotes can be used to produce nucleotide sequences, or their homologous polypeptides, proteins and peptides together with the embodiments. Commercially available and widely available systems include but are not limited to bacteria, mammals, yeast and insect cell systems. Different host cells have the characteristics and specific mechanisms of post-translational processing and modification of proteins. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the foreign proteins expressed. Those skilled in the art can use suitable expression system expression vectors to produce nucleotide sequences or their homologous polypeptides, proteins or peptides.

[0336] 【3. Methods of gene transfer】

[0337] Suitable methods for delivery of nucleic acids to affect expression of a composition are contemplated to include virtually any method for introducing nucleic acids (e.g., DNA, including viral and non-viral vectors) into a cell, tissue, or organism, as described herein or known to those of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference in its entirety), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference in its entirety); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference in its entirety); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippeet et al., 1993). al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microparticle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference in its entirety); by silicon carbide fiber agitation (Kaeppler et al., 1991); al., 1990; U.S. Patents 5,302,523 and 5,464,765, all of which are incorporated herein by reference in their entirety); by Agrobacterium-mediated transformation (U.S. Patents 5,591,616 and 5,563,055, all of which are incorporated herein by reference); or by PEG-mediated protoplast transformation (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, all of which are incorporated herein by reference in their entirety); by dehydration / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0338] 【4.Host cells】

[0339] In another aspect, it is considered to use a host cell that has been introduced into a recombinant expression vector. The antibody can be expressed in a variety of cell types. The expression construct encoding the antibody can be transfected into the cell according to a variety of methods known in the art. The vector DNA can be introduced into a prokaryotic or eukaryotic cell by conventional transformation or transfection techniques. Some vectors may use control sequences to enable it to replicate and / or express in prokaryotic and eukaryotic cells. In certain aspects, the antibody expression construct can be placed under the control of a promoter associated with T cell activation, such as a promoter controlled by NFAT-1 or NF-κΒ, both of which are transcription factors that can be activated when T cells are activated. The control of antibody expression enables T cells (such as tumor-targeted T cells) to sense their surrounding environment and regulate cytokine signaling in real time in the T cells themselves and the surrounding endogenous immune cells. Those skilled in the art will understand the conditions for incubating host cells to maintain them and allow vector replication. In addition, it is also understood and understood that the technology and conditions for allowing large-scale production of vectors and the production of nucleic acids encoded by vectors and their homologous polypeptides, proteins or peptides are also understood and understood.

[0340] For stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small fraction of cells can integrate the foreign DNA into their genome. In order to identify and select these integrants, selectable markers (e.g., for antibiotic resistance) are usually introduced into the host cells together with the target gene. Cells stably transfected with the introduced nucleic acid can be identified by drug selection and other methods known in the art (e.g., cells incorporating the selectable marker gene will survive, while other cells die).

[0341]

B.Separation

[0342] Nucleic acid molecules encoding the entire heavy and light chains of an antibody or one or both of their variable regions can be obtained from any source that produces the antibody. Methods for isolating mRNA encoding an antibody are well known in the art. For example, see Sambrook et al., supra. The sequences of human heavy and light chain constant region genes are also known in the art. For example, see Kabat et al., 1991, supra. Nucleic acid molecules encoding full-length heavy and / or light chains can be expressed in the introduced cells and the antibody isolated.

[0343]

II. Antibodies, antigen-binding fragments and peptides

[0344] The present disclosure includes any kind of antibody protein or polypeptide and any kind of chimeric protein or polypeptide molecule, including a chimeric protein or polypeptide molecule comprising part or all of the functionalities of an antibody protein or polypeptide. As used herein, "protein" or "polypeptide" refers to a molecule comprising at least five amino acid residues. The term "wild type" as used herein refers to an endogenous version of a molecule naturally present in an organism. In some embodiments, a wild type version of a protein or polypeptide is used, however, in many embodiments of the present disclosure, a modified protein or polypeptide is used to produce an immune response. The above terms can be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure (particularly its amino acid sequence) is changed relative to a wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide can have multiple activities or functions). It is particularly contemplated that a modified / variant protein or polypeptide may be changed relative to one activity or function, but retains a wild type activity or function in other aspects (e.g., immunogenicity). The term polypeptide also includes antibody fragments and antibody domains described herein, such as HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, HFRW1, HFRW2, HFRW3, HFRW4, LFRW1, LFRW2, LFRW3, LFRW4, V H 、V L , CH or CL.

[0345] When a protein is specifically mentioned herein, it generally refers to a natural (wild type) or recombinant (modified) protein, or optionally refers to a protein in which any signal sequence has been removed. The protein can be isolated directly from its natural organism, produced by recombinant DNA / exogenous expression methods, or produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In a specific embodiment, there are isolated nucleic acid fragments and recombinant vectors comprising nucleic acid sequences encoding polypeptides (e.g., antibodies or fragments thereof). The term "recombinant" can be used in conjunction with the name of a polypeptide or a specific polypeptide, which generally refers to a polypeptide produced by a nucleic acid molecule that has been manipulated in vitro or a replication product of such a molecule.

[0346] In certain embodiments, the size of the antibody, antigen binding fragment, protein or polypeptide (wild type or modified) can include, but is not limited to, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 400, 425 or 450 amino acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. It is contemplated that polypeptides may be mutated by truncation, rendering them shorter than the corresponding wild-type form, and furthermore, they may be altered by fusion or coupling to heterologous protein or polypeptide sequences having specific functions (e.g., for targeting or localization, for enhancing immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any distinct functional or structural unit of a protein or polypeptide, and generally refers to an amino acid sequence having a structure or function recognizable to one skilled in the art.

[0347] The antibodies, antigen binding fragments, polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins may include 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, or 50 (or any derivative range thereof) or more variant amino acids or nucleic acid substitutions, or may be substituted with SEQ ID NO: 4-27 is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) similar, identical or homologous to, 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69 ,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,12 6, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 2 17, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400 or more consecutive amino acids or nucleic acids, or any range derivable therein.

[0348] In some embodiments, the antibody, antigen-binding fragment, protein or polypeptide may comprise SEQ ID 4,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 1 45,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205 5, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347, 348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, (or any range thereof) of amino acids.

[0349] In some embodiments, the antibody, antigen-binding fragment or polypeptide may comprise SEQ ID NO: 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 1 45,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203,204,205 5, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307 07,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348 8,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389 , 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425 (or any derivative range thereof) of consecutive amino acids or nucleic acids. ,

[0350] In some embodiments, the antibody, antigen-binding fragment, protein or polypeptide may comprise SEQ ID NO: at least, at most, or exactly 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 7, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 1 43,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185,186,187,188,189,190,191,192,193,194,195,196,197,198,199,200,201,202,203 3,204,205,206,207,208,209,210,211,212,213,214,215,216,217,218,219,220,221,222,223,224,225,226,227,228,229,230,231,232,233,234,235,236,237,238,239,240,241,242,243,244,245,246,247,248,249,250,251,252,253,254,255,256,257,258,259,260,261,262,263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306 06,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,336,337,338,339,340,341,342,343,344,345,346,347,348 8,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379,380,381,382,383,384,385,386,387,388,389,390 , 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425 or more (or any derivative range thereof) of consecutive amino acids or nucleic acids, which are the same as SEQ IDNO: one of 4-9 or 22-27 is at least, at most or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein) similar, identical, or homologous.

[0351] In some aspects, there is a nucleic acid molecule, antibody, antigen-binding fragment, protein or polypeptide from any of positions 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,1 21, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169 9, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,

[0352] 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,

[0353] 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,

[0354] 246，247，248，249，250，251，252，253，254，255，256，257，258，259，

[0355] 260，261，262，263，264，265，266，267，268，269，270，271，272，273，

[0356] 274，275，276，277，278，279，280，281，282，283，284，285，286，287，

[0357] 288，289，290，291，292，293，294，295，296，297，298，299，300，301，

[0358] 302，303，304，305，306，307，308，309，310，311，312，313，314，315，

[0359] 316，317，318，319，320，321，322，323，324，325，326，327，328，329，

[0360] 330，331，332，333，334，335，336，337，338，339，340，341，342，343，

[0361] 344，345，346，347，348，349，350，351，352，353，354，355，356，357，

[0362] 358，359，360，361，362，363，364，365，366，367，368，369，370，371，

[0363] 372，373，374，375，376，377，378，379，380，381，382，383，384，385，

[0364] 386，387，388，389，390，391，392，393，394，395，396，397，398，399，

[0365] 400，401，402，403，404，405，406，407，408，409，410，411，412，413，

[0366] 414，415，416，417，418，419，420，421，422，423，424，425，426，427，

[0367] 428，429，430，431，432，433，434，435，436，437，438，439，440，441，

[0368] 442，443，444，445，446，447，448，449，450，451，452，453，454，455，

[0369] 456，457，458，459，460，461，462，463，464，465，466，467，468，469，

[0370] 470，471，472，473，474，475，476，477，478，479，480，481，482，483，

[0371] 484，485，486，487，488，489，490，491，492，493，494，495，496，497，

[0372] 498，499，500，501，502，503，504，505，506，507，508，509，510，511，

[0373] 512，513，514，515，516，517，518，519，520，521，522，523，524，525，

[0374] 526，527，528，529，530，531，532，533，534，535，536，537，538，539，

[0375] 540，541，542，543，544，545，546，547，548，549，550，551，552，553，

[0376] 554，555，556，557，558，559，560，561，562，563，564，565，566，567，

[0377] 568，569，570，571，572，573，574，575，576，577，578，579，580，581，

[0378] 582，583，584，585，586，587，588，589，590，591，592，593，594，595，

[0379] 596，597，598，599，600，601，602，603，604，605，606，607，608，609，

[0380] 610，611，612，613，614，615，616，617，618，619，620，621，622，623，

[0381] 624，625，626，627，628，629，630，631，632，633，634，635，636，637，

[0382] 638，639，640，641，642，643，644，645，646，647，648，649，650，651，

[0383] 652，653，654，655，656，657，658，659，660，661，662，663，664，665，

[0384] 666，667，668，669，670，671，672，673，674，675，676，677，678，679，

[0385] 680，681，682，683，684，685，686，687，688，689，690，691，692，693，

[0386] 694，695，696，697，698，699，700，701，702，703，704，705，706，707，

[0387] 708，709，710，711，712，713，714，715，716，717，718，719，720，721，

[0388] 722，723，724，725，726，727，728，729，730，731，732，733，734，735，

[0389] 736，737，738，739，740，741，742，743，744，745，746，747，748，749，

[0390] 750，751，752，753，754，755，756，757，758，759，760，761，762，763，

[0391] 764，765，766，767，768，769，770，771，772，773，774，775，776，777，

[0392] 778，779，780，781，782，783，784，785，786，787，788，789，790，791，

[0393] 792，793，794，795，796，797，798，799，800，801，802，803，804，805，

[0394] 806，807，808，809，810，811，812，813，814，815，816，817，818，819，

[0395] 820，821，822，823，824，825，826，827，828，829，830，831，832，833，

[0396] 834，835，836，837，838，839，840，841，842，843，844，845，846，847，

[0397] 848,849,850,851,852,853,854,855,856,857,858,859,860,861,862,863,864,865,866,867,868,869,870,871,872,873,874,875,876,877,878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910 10, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941 1,942,943,944,945,946,947,948,949,950,951,952,953,954,955,956,957,958,959,960,961,962,963,964,965,966,967,968,969,970,971,972 , 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999 or 1000, including at least, at most or Okay 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, 46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87 ,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122，123，124，125，126，127，128，129，130，131，132，133，134，135，136，137，138，139，140，141，142，143，144，145，146，147，148，149，150，151，152，153，154，155，156，157，158，159，160，161，162，163，164，165，166，167，168，169，170，171，172，173，174，175，176，177，178，179，180，181，182，183，184，185，186，187，188，189，190，191，192，193，194，195，196，197，198，199，200，201，202，203，204，205，206，207，208，209，210，211，212，213，214，215，216，217，218，219，220，221，222，223，224，225，226，227，228，229，230，231，232，233，234，235，236，237，238，239，240，241，242，243，244，245，246，247，248，249，250，251，252，253，254，255，256，257，258，259，260，261，262，263，264，265，266，267，268，269，270，271，272，273，274，275，276，277，278，279，280，281，282，283，284，285，286，287，288，289，290，291，292，293，294，295，296，297，298，299，300，301，302，303，304，305，306，307，308，309，310，311，312，313，314，315，316，317，318，319，320，321，322，323，324，325，326，327，328，329，330，331，332，333，334，335，336，337，338，339，340，341，342，343，344，345，346，347，348，349，350，351，352，353，354，355，356，357，358，359，360，361，362，363，364，365，366，367，368，369，370，371，372，373，374，375，376，377，378，379，380，381，382，383，384，385，386，387，388，389，390，391，392，393，394，395，396，397，398，399，400，401，402，403，404，405，406，407，408，409，410，411，412，413，414，415，416，417，418，419，420，421，422，423，424，425，426，427，428，429，430，431，432，433，434，435，436，437，438，439，440，441，442，443，444，445，446，447，448，449，450，451，452，453，454，455，456，457，458，459，460，461，462，463，464，465，466，467，468，469，470，471，472，473，474，475，476，477，478，479，480，481，482，483，484，485，486，487，488，489，490，491，492，493，494，495，496，497，498，499，500，501，502，503，504，505，506，507，508，509，510，511，512，513，514，515，516，517，518，519，520，521，522，523，524，525，526，527，528，529，530，531，532，533，534，535，536，537，538，539，540，541，542，543，544，545，546，547，548，549，550，551，552，553，554，555，556，557，558，559，560，561，562，563，564，565，566，567，568，569，570，571，572，573，574，575，576，577，578，579，580，581，582，583，584，585，586，587，588，589，590，591，592，593，594，595，596，597，598，599，600，601，602，603，604，605，606，607，608，609，610，611，612，613，614，615，616，617，618，619，620，621，622，623，624，625，626，627，628，629，630，631，632，633，634，635，636，637，638，639，640，641，642，643，644，645，646，647，648，649，650，651，652，653，654，655，656，657，658，659，660，661，662，663，664，665，666，667，668，669，670，671，672，673，674，675，676，677，678，679，680，681，682，683，684，685，686，687，688，689，690，691，692，693，694，695，696，697，698，699，700，701，702，703，704，705，706，707，708，709，710，711，712，713，714，715，716，717，718，719，720，721，722，723，724，725，726，727，728，729，730，731，732，733，734，735，736，737，738，739，740，741，742，743，744，745，746，747，748，749，750，751，752，753，754，755，756，757，758，759，760，761，762，763，764，765，766，767，768，769，770，771，772，773，774，775，776，777，778，779，780，781，782，783，784，785，786，787，788，789，790，791，792，793，794，795，796，797，798，799，800，801，802，803，804，805，806，807，808，809，810，811，812，813，814，815，816，817，818，819，820，821，822，823，824，825，826，827，828，829，830，831，832，833，834，835，836，837，838，839，840，841，842，843，844，845，846，847，848，849，850，851，852，853，854，855，856，857，858，859，860，861，862，863，864，865，866，867，868，869，870，871，872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905 05, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 93 8,939,940,941,942,943,944,945,946,947,948,949,950,951,952,953,954,955,956,957,958,959,960,961,962,963,964,965,966,967,968,969,970,971 , 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999 or 1000 (or any derivable range therein) any consecutive amino acids or nucleotides in SEQ ID NO: 4 to 27. ,

[0398] In some embodiments, the amino acid is at position 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 ,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,9 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,1 55, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186 6, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 , 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292 2,293,294,295,296,297,298,299,300,301,302,303,304,305,306,307,308,309,310,311,312,313,314,315,316,317,318,319,320,321,322,323,324,325,326,327,328,329,330,331,332,333,334,335,3 36,337,338,339,340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358,359,360,361,362,363,364,365,366,367,368,369,370,371,372,373,374,375,376,377,378,379 , 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399 or 400 is substituted with alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. ,

[0399] Nucleotide and protein, polypeptide and peptide sequences of various genes have been previously disclosed and can be found in recognized computerized databases. Two commonly used databases are the Genbank and GenPept databases of the National Center for Biotechnology Information (at www.ncbi.nlm.nih.gov / ) and the Universal Protein Resource (UniProt; at www.uniprot.org). The coding regions of these genes can be amplified and / or expressed using the techniques disclosed herein or techniques known to those of ordinary skill in the art.

[0400] It is contemplated that in the compositions of the present disclosure, about 0.001 mg to about 10 mg of total polypeptides, peptides and / or proteins are present per milliliter. The concentration of protein in the composition can be about, at least about, or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg / ml or more (or any range derivable therein).

[0401] [V. Sequence]

[0402] Embodiments of polypeptides, antibodies, and antigen-binding fragments are contemplated herein.

[0403] The following is a discussion about changing the amino acid subunits of a protein to produce an equivalent or even improved second generation variant polypeptide or peptide. For example, certain amino acids can replace other amino acids in a protein or polypeptide sequence, regardless of whether the ability to interact with a structure (e.g., an antigen binding region of an antibody or a binding site on a substrate molecule) is significantly lost. Since the interaction ability and properties of a protein determine the functional activity of the protein, certain amino acid substitutions can be made in a protein sequence and its corresponding DNA coding sequence, and still produce a protein with similar or desirable properties. Therefore, the inventors envision that the DNA sequence of a gene encoding a protein may undergo various changes without significantly losing its biological utility or activity.

[0404] The term "functionally equivalent codons" is used herein to refer to codons encoding the same amino acid, such as six different codons for arginine. Also contemplated are "neutral substitutions" or "neutral mutations," which refer to changes in one or more codons encoding biologically equivalent amino acids.

[0405] The amino acid sequence variants disclosed herein can be substitution, insertion or deletion variants. The changes in the polypeptides disclosed herein may affect 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, 50 or more non-continuous or continuous amino acids of the protein or polypeptide, compared to the wild type. The variant may comprise an amino acid sequence that is at least 50%, 60%, 70%, 80% or 90% identical to any sequence provided or cited herein, including all values ​​and ranges therebetween. The variant may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more substituted amino acids.

[0406] It is also understood that amino acid and nucleic acid sequences may include added residues, such as an added N-terminal or C-terminal amino acid, or a 5' or 3' sequence, respectively, but still be substantially identical to one of the sequences disclosed herein, provided that the sequence meets the criteria specified above, including maintaining biological protein activity in cases involving protein expression. Adding terminal sequences is particularly useful for nucleic acid sequences, for example, and may include various non-coding sequences on either side of the 5' or 3' portion of the coding region.

[0407] Deletion variants generally lack one or more residues of the native or wild-type protein. Single residues may be deleted, or multiple consecutive amino acids may be deleted. Stop codons may be introduced (by substitution or insertion) into the encoding nucleic acid sequence to produce truncated proteins.

[0408] Insertion mutants generally involve the addition of amino acid residues at non-terminal points of a polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be produced and may include fusion proteins that are multimers or concatemers of one or more peptides or polypeptides described or referenced herein.

[0409] Substitution variants typically involve exchanging one amino acid for another at one or more sites within a protein or polypeptide, and can be designed to modulate one or more properties of a polypeptide, with or without loss of other functions or properties. Substitutions may be conservative, i.e., replacing one amino acid with an amino acid having similar chemical properties. "Conservative amino acid substitutions" may involve exchanging a member of an amino acid class for another member of the same class. Conservative substitutions are well known in the art and include, for example, changes such as alanine to serine, arginine to lysine, asparagine to glutamine or histidine, aspartic acid to glutamic acid, cysteine ​​to serine, glutamine to asparagine, glutamic acid to aspartic acid, glycine to proline, histidine to asparagine or glutamine, isoleucine to leucine or valine, leucine to valine or isoleucine, lysine to arginine, methionine to leucine or isoleucine, phenylalanine to tyrosine, leucine or methionine, serine to threonine, threonine to serine, tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, valine to isoleucine or leucine. Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. These include other reverse or inverted forms of peptidomimetics or amino acid moieties.

[0410] Alternatively, a substitution may be "non-conservative", such that the function or activity of the polypeptide is affected. Non-conservative changes generally involve replacing an amino acid residue with an amino acid residue of a different chemical nature, such as replacing a non-polar or uncharged amino acid with a polar or charged amino acid, or vice versa. Non-conservative substitutions may involve exchanging a member of one amino acid class for a member of another class.

[0411] Those skilled in the art can use known techniques to determine suitable variants of polypeptides as described herein. Those skilled in the art can identify suitable regions of molecules by targeting regions that are considered unimportant to activity, and these regions can be changed without destroying activity. The skilled artisan will also be able to identify conservative portions and amino acid residues of molecules in similar proteins or polypeptides. In further embodiments, conservative amino acid substitutions may be made in regions that may be important for biological activity or structure without significantly changing biological activity or adversely affecting protein or polypeptide structure.

[0412] When making such changes, the hydropathicity index of the amino acids can be considered. The hydropathicity profile of a protein is calculated by assigning a numerical value (the "hydropathy index") to each amino acid and then averaging these values ​​repeatedly along the peptide chain. Each amino acid is assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al., J. Mol. Biol. 157:105-131 (1982)). It is generally accepted that the relative hydrophobicity of amino acids contributes to the secondary structure of the resulting protein or polypeptide, which in turn defines the interaction of the protein or polypeptide with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. It is well known that certain amino acids can be substituted with other amino acids having a similar hydropathic index or score and still retain similar biological activity. When making changes based on the hydropathic index, in certain embodiments, substitutions of amino acids with hydropathic indices within ±2 are included. In certain aspects of the invention, those within the range of ±1 are included, while in other aspects of the invention, those within the range of ±0.5 are included.

[0413] It is also understood in the art that substitution of similar amino acids can be effectively achieved based on hydrophilicity. U.S. Pat. No. 4,554,101 (incorporated herein by reference in its entirety) indicates that the maximum local average hydrophilicity of a protein (controlled by the hydrophilicity of its adjacent amino acids) is related to the biological properties of the protein. In certain embodiments, the maximum local average hydrophilicity of a protein, as controlled by the hydrophilicity of its adjacent amino acids, is related to its immunogenicity and antigen binding, i.e., as a biological property of the protein. The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). When making changes based on similar hydrophilicity values, in certain embodiments, substitution of amino acids with hydrophilicity values ​​within ±2 is included, in other embodiments, amino acids with hydrophilicity values ​​within ±1 are included, and in other embodiments, amino acids within ±0.5 are included. In some cases, epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These regions are also referred to as "epitope core regions." It is understood that an amino acid can be substituted with another amino acid having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0414] In addition, one skilled in the art can review structure-function studies to identify residues in similar polypeptides or proteins that are important for activity or structure. In view of this comparison, the importance of amino acid residues in proteins can be predicted, which residues correspond to amino acid residues in similar proteins that are important for activity or structure. One skilled in the art can select chemically similar amino acids to replace the important amino acid residues for such predictions.

[0415] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence of the related structure in similar proteins or polypeptides. In view of this information, those skilled in the art can predict the arrangement of the amino acid residues of the antibody relative to its three-dimensional structure. Those skilled in the art can choose not to change the amino acid residues predicted to be located on the surface of the protein, because these residues may be involved in important interactions with other molecules. In addition, those skilled in the art can generate test variants comprising single amino acid substitutions at each desired amino acid residue. Standard assays can then be used to screen the binding and / or activity of these variants, thereby generating information collected from such routine experiments, which may allow those skilled in the art to determine the amino acid positions that should be avoided from further substitutions alone or in combination with other mutations. Various tools that can be used to determine secondary structure can be found at www.expasy.org / proteomics / protein_structure.

[0416] In some embodiments of the invention, amino acid substitutions are made to: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand or antigen binding affinity, and / or (5) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made according to the naturally occurring sequence. Substitutions can be made in portions of the antibody that are located outside of the domains that form intermolecular contacts. In such embodiments, conservative amino acid substitutions that do not significantly change the structural characteristics of the protein or polypeptide can be used (e.g., one or more substituted amino acids that do not disrupt the secondary structure that characterizes the native antibody).

[0417]

III. Nucleic Acids

[0418] In certain embodiments, the nucleic acid sequence may be present in a variety of examples, such as: recombinant vectors and isolated fragments incorporating the sequence or recombinant polynucleotides encoding the peptides and polypeptides disclosed herein, or fragments, derivatives, mutant proteins or variants thereof, polynucleotides sufficient to be used as hybridization probes, PCR primers or sequencing primers to identify, analyze, mutate or amplify polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting polynucleotide expression, and the above-mentioned complementary sequences described herein. Nucleic acids encoding fusion proteins comprising these peptides are also provided. Nucleic acids may be single-stranded or double-stranded, and may contain RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0419] The term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, such as plasmids, cosmids, phages, viruses, etc. Polynucleotides include regulatory sequences in some aspects, which are essentially separated from their naturally occurring genes or protein coding sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences can, but need not, be present in a polynucleotide.

[0420] In this regard, the term "gene", "polynucleotide" or "nucleic acid" is used to refer to a nucleic acid encoding a protein, polypeptide or peptide (including any sequences required for proper transcription, post-translational modification or localization). As will be appreciated by those skilled in the art, the term includes genomic sequences, expression cassettes, cDNA sequences and smaller engineered nucleic acid fragments that express or may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins and mutants. A nucleic acid encoding all or part of a polypeptide may comprise a continuous nucleic acid sequence encoding all or part of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid containing variants that have slightly different nucleic acid sequences, but nonetheless encode the same or substantially similar proteins.

[0421] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; compared to the polynucleotide sequences provided herein, comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity, including all values ​​in between, using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90%, preferably 95% and above identity to the amino acid sequence described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide.

[0422] Nucleic acid fragments, regardless of the length of the coding sequence itself, can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction endonuclease sites, multiple cloning sites, other coding fragments, etc., so that their overall length can vary greatly. The nucleic acids can be of any length. For example, they can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can contain one or more additional sequences, such as regulatory sequences, and / or be part of a larger nucleic acid, such as a vector. Thus, it is contemplated that nucleic acid fragments of virtually any length can be used, with the overall length preferably being limited by ease of preparation and use in the intended recombinant nucleic acid scheme. In some cases, the nucleic acid sequence may encode a polypeptide sequence that carries an additional heterologous coding sequence, for example, to allow purification, transport, secretion, post-translational modification of the polypeptide, or to achieve therapeutic benefits such as targeting or therapeutic efficacy. As described above, a tag or other heterologous polypeptide can be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is different from the modified polypeptide.

[0423] The change can be introduced into the nucleic acid by mutation, thereby causing the amino acid sequence of the polypeptide (for example antigenic peptide or polypeptide) encoded by it to change. Any technology known in the art can be used to import mutation. In one embodiment, use for example site-directed mutagenesis scheme to change one or more specific amino acid residues. In another embodiment, use for example random mutagenesis scheme to change one or more randomly selected residues. In any case, the mutant polypeptide can express and screen out required characteristics.

[0424] Mutations can be introduced into a nucleic acid without significantly changing the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made, resulting in amino acid substitutions of non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid to selectively change the biological activity of the polypeptide it encodes. See Romain Studer et al., Biochem. J. 449: 581-594 (2013). For example, mutations can quantitatively or qualitatively change biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating activity. Examples of qualitative changes include changing the antigenic specificity of an antibody.

[0425] [VI. Peptide expression]

[0426] In some aspects, there are nucleic acid molecules encoding polypeptides, antibodies or antigen binding fragments of the present disclosure. Nucleic acid molecules can be used to express a large number of polypeptides, including antibodies and / or chimeric polypeptides using them. If the nucleic acid molecules are derived from non-human, non-transgenic animals, the nucleic acid molecules can be used for humanization of antibody or TCR genes.

[0427] A. Carrier

[0428] In some aspects, the envisioned expression vector comprises a nucleic acid molecule encoding a polypeptide of a desired sequence or portion thereof (e.g., a fragment comprising one or more CDRs or one or more variable region domains). An expression vector comprising a nucleic acid molecule may encode a heavy chain, a light chain, or an antigen binding portion thereof. In some aspects, an expression vector comprising a nucleic acid molecule may encode a fusion protein, a modified antibody, an antibody heavy chain and / or light chain, an antibody fragment, and a probe thereof. In addition to control sequences for controlling transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0429] To express the disclosed polypeptides or peptides, the DNA encoding the polypeptides or peptides is inserted into an expression vector so that the gene region is operably linked to transcriptional and translational control sequences. In certain aspects, the vector encoding a fully functional human CH or CL immunoglobulin sequence has appropriate restriction sites so that any V H or V L Sequence. In some aspects, the vector encoding a fully functional human TCRα or TCRβ sequence has a sequence of appropriate restriction sites, so that any variable sequence or CDR1, CDR2 and / or CDR3 can be easily inserted and expressed. Generally, the expression vector used in any host cell contains sequences for plasmid or virus maintenance and cloning and expression of exogenous nucleotide sequences. Such sequences are collectively referred to as "flanking sequences" and generally include one or more of the following operably connected nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a polypeptide secretion leader sequence, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Such sequences and methods of using them are well known in the art.

[0430]

B. Expression system

[0431] There are many expression systems, which at least comprise a part or all of the above-mentioned expression vectors. Systems based on prokaryotes and / or eukaryotes can be used for use with embodiments to produce nucleotide sequences, or their homologous polypeptides, proteins and peptides. Commercially available and widely available systems include but are not limited to bacteria, mammals, yeast and insect cell systems. Different host cells have the characteristics and specific mechanisms of post-translational processing and modification of proteins. Suitable cell lines or host systems can be selected to ensure correct modification and processing of the foreign proteins expressed. Those skilled in the art can use suitable expression system expression vectors to produce nucleotide sequences or their homologous polypeptides, proteins or peptides.

[0432]

C. Methods of gene transfer

[0433] Suitable methods for delivery of nucleic acids to affect expression of a composition are contemplated to include virtually any method for introducing nucleic acids (e.g., DNA, including viral and non-viral vectors) into cells, tissues, or organisms, as described herein or known to those of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA, such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference in its entirety), including microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference in its entirety); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference in its entirety); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1993); et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonication (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microparticle bombardment (PCT Application Nos. WO 94 / 09699 and 95 / 06128; U.S. Pat. Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference in its entirety); by silicon carbide fiber agitation (Kaeppler et al., 1991). al., 1990; U.S. Patents 5,302,523 and 5,464,765, all of which are incorporated herein by reference in their entirety); by Agrobacterium-mediated transformation (U.S. Patents 5,591,616 and 5,563,055, all of which are incorporated herein by reference); or by PEG-mediated protoplast transformation (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, all of which are incorporated herein by reference in their entirety); by dehydration / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0434]

VII. Treatment methods

[0435] In various embodiments, diseased cells or other cells expressing TdT peptides on their surface are targeted for the purpose of improving the medical condition in an individual with the medical condition, or for the purpose of reducing the risk of or delaying the severity and / or onset of the medical condition in an individual. In certain instances, cancer cells expressing endogenous TdT are targeted for the purpose of killing the cancer cells.

[0436] The target TdT peptide antibody constructs, nucleic acid sequences, vectors, immune cell expression are the same as those contemplated herein, and / or contain the same pharmaceutical compositions for preventing, treating or ameliorating cancerous diseases, such as tumor diseases. In specific embodiments, the pharmaceutical compositions disclosed herein may be particularly suitable for preventing, ameliorating and / or treating cancers, including cancers expressing TdT peptides and cancers that may or may not be, for example, solid tumors.

[0437] Administration of the disclosed compositions is useful for all stages (I, II, III, or IV) and types of cancer, including, for example, minimal residual disease, early stage cancer, advanced stage cancer, and / or metastatic cancer and / or refractory cancer.

[0438] The disclosure also includes co-administration schemes with other compounds, such as surgery, radiotherapy, chemotherapy, other immunotherapy, drug therapy or hormone therapy, or combinations thereof. Clinical schemes for co-administration of inventive compounds may include co-administration before or after the administration of another component, or simultaneously. Specific combination therapies include chemotherapy, radiotherapy, surgery, hormone therapy, or other types of immunotherapy.

[0439] VIII. Evaluation, testing and diagnostic methods

[0440] Embodiments of the present disclosure include methods for evaluating any sample from a subject, the method comprising contacting a biological sample from a subject or an extract thereof with at least one antibody, antigen binding fragment, or polypeptide contained herein. In various embodiments, a method for detecting cancer is provided, comprising contacting a biological sample from a subject or an extract thereof with at least one antibody, antigen binding fragment, or polypeptide contained herein. In a specific embodiment, there is a method for diagnosing cancer in a subject, the method comprising contacting a biological sample from a subject or an extract thereof with at least one antibody, antigen binding fragment, or polypeptide. In any evaluation method, the sample can be of any kind, but in a specific embodiment, it includes a blood sample, a urine sample, a stool sample, a nasopharyngeal sample, a cerebrospinal fluid sample, a cheek scrape sample, a nipple aspiration sample, a biopsy sample, or a combination thereof.

[0441] In various embodiments for evaluation, detection or diagnosis, at least one antibody, Fab or polypeptide is operably linked to a detectable marker. Any method may also include incubating the antibody, Fab or polypeptide under conditions that allow the antibody, Fab or polypeptide to bind to an antigen in a biological sample or an extract thereof. The method may further include detecting the binding of the antigen to the antibody, Fab or polypeptide. The method may also include contacting the biological sample with at least one capture antibody, antigen or polypeptide. The capture antibody may be linked to a solid support.

[0442] In a specific embodiment, at least one antibody, Fab or polypeptide is operably linked to a detectable marker. The method may also include incubating the antibody, Fab or polypeptide under conditions that allow the antibody, Fab or polypeptide to bind to an antigen in the biological sample or an extract thereof. In various embodiments, the method also includes detecting the binding of the antigen to the antibody, Fab or polypeptide. The method may also include contacting the biological sample with at least one capture antibody, antigen or polypeptide.

[0443] [IX. Pharmaceutical composition]

[0444] The present disclosure includes methods for treating disease and modulating immune responses in subjects in need thereof. The disclosure includes cells, which may be in the form of pharmaceutical compositions, that can be used to induce or alter an immune response.

[0445] Administration according to the currently disclosed compositions will generally be carried out by any conventional route. This includes, but is not limited to, parenteral, in situ, intradermal, subcutaneous, oral, transdermal, intramuscular, intraperitoneal, intraorbital, implant, inhalation, intraventricular, intranasal, intraarterial, intratracheal, intrapleural, intratumoral, endoscopic, intralesional, intracranial, transdermal, regional, systemic, perfusion, tumor microenvironment, or intravenous injection. In some embodiments, the compositions of the present disclosure (e.g., compositions comprising TdT peptide-binding polypeptides) are administered intravenously to a subject.

[0446] Typically, the compositions and therapies disclosed herein are administered in a manner compatible with the dosage formulation, and in an amount that is therapeutically effective and immunomodifying. The amount administered depends on the subject to be treated. The exact amount of active ingredient that needs to be administered depends on the judgment of the practitioner.

[0447] The mode of application can vary widely. Any conventional method for administering a pharmaceutical composition comprising a cellular component is suitable. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject.

[0448] In many cases, it is best to have up to or at least 3, 4, 5, 6, 7, 8, 9, 10 or more multiple administrations. The interval between administrations is 2 days to 12 weeks, usually 1 to 2 weeks.

[0449] The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active ingredient, they are contemplated for use in immunogenic and therapeutic compositions. The pharmaceutical compositions disclosed herein are pharmaceutically acceptable compositions.

[0450] The compositions of the present disclosure can be formulated for parenteral administration, for example, for injection by intravenous, intramuscular, subcutaneous or even intraperitoneal routes. Generally, such compositions can be prepared as injections, can be liquid solutions or suspensions, and the preparations can also be emulsified.

[0451] The drug form suitable for injectable use includes sterile aqueous solutions or dispersions; formulations include sesame oil, peanut oil or propylene glycol in water. It should also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0452] The preparation method of sterile injection solution is to mix the required amount of active ingredient (e.g., polypeptide of the present disclosure) into an appropriate solvent, and as required, mix the above-mentioned various other ingredients, and then filter sterilize. Generally, dispersions are prepared by mixing various sterilized active ingredients into sterile carriers, which contain basic dispersion medium and other ingredients required by the above-mentioned ingredients.

[0453] The effective amount of the composition is determined based on the intended target. The term "unit dose" or "dose" refers to a physical discrete unit suitable for use in a subject, each unit containing a predetermined number of compositions, calculated to produce the desired response associated with its administration (i.e., appropriate route and regimen). Depending on the number of treatments and the unit dose, the amount to be administered depends on the desired result and / or protection. The precise amount of the composition also depends on the practitioner's judgment and is unique to each person. Factors affecting dosage include the physical and clinical state of the subject, route of administration, intended therapeutic goal (symptom relief and cure), and the efficacy, stability, and toxicity of the specific composition. After preparation, the solution will be administered in a manner compatible with the dosage formulation and administered in an amount effective for treatment or prevention. These preparations are easily administered in various dosage forms, such as the type of injection solution described above.

[0454] The disclosed compositions and related methods, in particular the administration of the disclosed compositions, can also be used in conjunction with the administration of other therapies, such as the additional therapies described herein or in combination with other conventional therapies known in the art.

[0455] The therapeutic compositions and treatments disclosed herein can precede, be concurrent with, and / or be subsequent to another treatment or drug, with intervals ranging from a few minutes to a few weeks. In embodiments where the agents are applied separately to cells, tissues, or organisms, it will generally be ensured that there is no considerable time between the times of each delivery for the therapeutic agents to expire, so that the therapeutic agents can still produce a favorable combined effect on the cells, tissues, or organisms. For example, in this case, it is conceivable that a person can contact cells, tissues, or organisms with two, three, four, or more agents or treatments almost simultaneously (i.e., in less than a minute). In other aspects, the time period may be 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours , 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks or longer, and any range derivable therein, to administer or provide one or more agents or treatments before and / or after administration of another agent or treatment.

[0456] The treatment may include various "unit doses". A unit dose is defined as a therapeutic composition containing a predetermined amount. The amount to be administered, as well as the specific route and formulation, are within the discretion of the clinical practitioner. A unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. In some embodiments, a unit dose includes a single administrable dose.

[0457] The amount to be administered depends on both the number of treatments and the unit dose, and the desired therapeutic effect. An effective dose refers to the amount required to achieve a specific effect. In the practice of certain embodiments, it is contemplated that doses within the range of 10 mg / kg to 200 mg / kg may affect the protective ability of these agents. Thus, it is estimated that dosages include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day or mg / day or any range derived therefrom. In addition, such dosages can be administered multiple times in one day, and / or administered over multiple days, weeks or months.

[0458] In some embodiments, the therapeutically effective or sufficient amount of antibody administered to a human body will be in the range of about 0.01 to about 50 mg / kg of the patient's body weight, whether by one or more administrations. In some embodiments, the treatment used is, for example, about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg administered daily. In one embodiment, the therapy described herein is administered to a subject at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of a 21-day cycle. The dose can be administered as a single dose or multiple doses (e.g., 2 or 3 doses), such as by infusion. The progress of this therapy is easily monitored by conventional techniques.

[0459] In certain embodiments, the effective dose of the pharmaceutical composition is a dose that provides a blood level of about 1 μM to 150 μM. In another embodiment, the effective dose provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derived therefrom). In other embodiments, the dose can provide a blood level of the therapeutic agent resulting from administration of the therapeutic agent to a subject of about, at least, or at most about 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 μM or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in vivo to a metabolic therapeutic agent, in which case the blood level may refer to the amount of the agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels discussed herein may refer to the unmetabolized therapeutic agent.

[0460] The exact amount of the therapeutic composition also depends on the judgment of the practitioner and is unique to each individual. Factors affecting dosage include the patient's physical and clinical state, route of administration, intended therapeutic goal (relief of symptoms vs. cure), and other therapies the subject may be receiving or the efficacy, stability, and toxicity of the specific therapeutic substance.

[0461] Those skilled in the art will be able to understand and appreciate that dosage units of μg / kg or mg / kg body weight can be converted and expressed as similar concentration units of μg / ml or mM (blood levels), such as 4 μM to 100 μM. It should also be understood that uptake depends on the species and organ / tissue. Applicable conversion factors and physiological assumptions to be made about uptake and concentration measurements are well known and will allow those skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions about the dosages, effects and results described herein.

[0462]

X. Detectable tags

[0463] In certain aspects of the present disclosure, it will be useful to detect or therapeutically label any type of antibody, including at least one Fab polypeptide or protein G Fab binding domain. Methods for coupling polypeptides to these reagents are known in the art. For illustrative purposes only, polypeptides can be labeled with detectable moieties (such as radioactive atoms, chromophores, fluorophores, etc.). Such labeled polypeptides can be used in diagnostic techniques, either in vivo, in isolated test samples, or in the methods described herein.

[0464] As used herein, the term "label" means a directly or indirectly detectable compound or composition, which is directly or indirectly coupled to a composition to be detected, such as a polynucleotide or protein, such as an antibody, to produce a "labeled" composition. The term also includes sequences coupled to polynucleotides that provide signals when the inserted sequence is expressed, such as green fluorescent protein (GFP), etc. The label itself may be detectable (e.g., radioisotope labels or fluorescent labels), or in the case of enzyme labels, it may catalyze chemical changes in detectable substrate compounds or compositions. These labels may be suitable for small-scale detection, or more suitable for high-throughput screening. Therefore, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and proteins, including enzymes. Labels can be simply detected or quantified. A simply detected response typically includes a response that is only confirmed to exist, while a quantified response typically includes a response with a quantifiable (e.g., digitally reportable) value, such as intensity, polarization and / or other attributes. In luminescent or fluorescent assays, the detectable response can be produced directly using a luminophore or fluorophore associated with the assay component that actually participates in binding, or indirectly using a luminophore or fluorophore associated with another (eg, reporter or indicator) component.

[0465] Examples of luminescent markers that generate signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescent responses generally include changes or occurrences of luminescent signals. Suitable methods and luminophores for measuring components of luminescent markers known in the art are described, for example, in Haugland, Richard P., (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0466] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrin, coumarin, methylcoumarin, pyrene, malate green, stilbene, fluorescent yellow, sulfonated pyrene, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).

[0467] In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present on a cell or tissue surface or on a surface, such as a cell surface marker. Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimide groups, succinimide esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule. The choice of the fluorescent label functional group will depend on the site of attachment to the linker, reagent, label, or second labeling agent.

[0468] Attachment of the fluorescent label can be direct attachment to the cellular component or compound, or can be through a linker. Suitable binding pairs for indirect attachment of the fluorescent label to the intermediate include, but are not limited to, antigen / polypeptide, such as rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0469] Coupling of a polypeptide to a low molecular weight hapten can increase the sensitivity of the antibody in the assay. The hapten can then be specifically detected by a second reaction. For example, haptens such as biotin are commonly used, which react with avidin, or dinitrophenol, pyridoxal and fluorescein, which react with specific anti-hapten polypeptides. See Harlow and Lane (1988), supra.

[0470]

XI. Sample preparation

[0471] In some aspects, the method involves obtaining or evaluating a sample from a subject using the antibodies and antibody-related reagents contained herein. The sample may include a sample obtained from any source, including but not limited to blood, sweat, hair follicles, oral tissue, tears, menstruation, feces, aspirates, cerebrospinal fluid, sputum, cheek scrapings and / or saliva. In some aspects of the current method, any medical professional, such as a doctor, nurse, or medical technician, can obtain a biological sample for testing. In addition, the biological sample can be obtained without the help of a medical professional. The sample can be obtained by the individual who needs the sample analysis.

[0472] The sample may include, but is not limited to, tissues, cells, or biological materials from or derived from cells of a subject. A biological sample may be a heterogeneous or homogeneous population of cells or tissues. A biological sample may be obtained using any method known in the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods, including, but not limited to: scraping the skin or cervix, swabbing the cheek, saliva collection, urine collection, stool collection, menstrual collection, tears, or semen collection.

[0473] The sample can be obtained by methods known in the art. In some embodiments, the sample is obtained by biopsy. In other embodiments, the sample is obtained by swab, endoscopy, scraping, phlebotomy or any other method known in the art. In some cases, the components of the kit of the current method can be used to obtain, store or transport samples. In some cases, multiple samples can be obtained by the method described herein, such as multiple esophageal samples for diagnosis. In other cases, multiple samples can be obtained by these methods, such as one or more samples from a type of tissue (such as esophagus) and one or more samples from another sample (such as serum) for diagnosis. In some cases, multiple samples may be obtained at the same or different times, such as one or more samples from a type of tissue (such as esophagus) and one or more samples from another sample (such as serum). Samples may be obtained at different times, stored and / or analyzed by different methods. For example, samples can be obtained and analyzed by conventional staining methods or any other cytological analysis methods.

[0474] In some embodiments, the biological sample can be obtained by a physician, nurse, or other medical professional, such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or pulmonologist. The medical professional may instruct appropriate tests or assays to be performed on the sample. In some aspects, the molecular analysis business may consult which tests or assays are most appropriate. In other aspects of the current method, the patient or subject can obtain a biological sample for testing without the assistance of a medical professional, such as obtaining a blood sample, a urine sample, a stool sample, a nasopharyngeal sample, a cerebrospinal fluid sample, a cheek scrape sample, a nipple aspiration sample, a biopsy sample, or a combination thereof.

[0475] In other cases, the sample is obtained by an invasive procedure, including but not limited to: biopsy, needle aspiration, endoscopy or phlebotomy. Needle aspiration methods may further include fine needle puncture, core needle biopsy, vacuum-assisted biopsy or large core biopsy. In some embodiments, multiple samples can be obtained by the methods herein to ensure sufficient amounts of biological material.

[0476] The general method of obtaining biological samples is also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001 (incorporated herein in their entirety as reference) describe the general method and cytological methods of biopsy. In one embodiment, the sample is a fine needle aspirate of an esophageal or suspected esophageal tumor or tumor. In some cases, the fine needle aspiration sampling procedure can be guided by the use of ultrasound, X-ray or other imaging equipment.

[0477] In some embodiments of the present method, the molecular analysis company may obtain the biological sample directly from the subject, or from a medical professional, a third party, or a kit provided by the molecular analysis company or a third party. In some cases, the biological sample may be obtained by the molecular analysis company after the subject, medical professional, or third party obtains the biological sample and sends it to the molecular analysis company. In some cases, the molecular analysis company may provide suitable containers and excipients for storing and transporting the biological sample to the molecular analysis company.

[0478] In some embodiments of the methods described herein, a medical professional does not need to participate in the preliminary diagnosis or sample collection. Individuals can also obtain samples by using an over-the-counter (OTC) kit. The OTC kit may include a method for obtaining the sample described herein, a method for storing the sample for inspection, and instructions for the correct use of the kit. In some cases, molecular analysis services are included in the purchase price of the kit. In other cases, molecular analysis services are billed separately. Samples suitable for use in molecular analysis services can be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of the individual to be tested. A method for determining sample suitability and / or adequacy is provided.

[0479] In some embodiments, the subject may be sent to a specialist such as an oncologist, surgeon, or endocrinologist. The specialist may also obtain the biological sample for testing, or send the individual to a testing center or laboratory to submit the biological sample. In some cases, a medical professional may send the subject to a testing center or laboratory to submit the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular analysis business may obtain the sample.

[0480] IV. Host cells

[0481] As used herein, the terms "cell", "cell line" and "cell culture" can be used interchangeably. All of these terms also include freshly isolated cells and cells cultured in vitro, activated or amplified. All of these terms also include their offspring, i.e., any and all subsequent generations. It is understood that all offspring may not be identical due to intentional or unintentional mutations. In the context of expressing heterologous nucleic acid sequences, "host cell" refers to a prokaryotic or eukaryotic cell, which includes any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by a vector. Host cells can and have been used as receptors for vectors or viruses. Host cells can be "transfected" or "transformed," which refers to the process by which exogenous nucleic acids (such as recombinant protein coding sequences) are transferred or introduced into host cells. Transformed cells include primary subject cells and their offspring.

[0482] In certain embodiments, transfection can be performed on any prokaryotic or eukaryotic cell. In certain aspects, electroporation relates to transfection of human cells. In other aspects, electroporation relates to transfection of animal cells. In certain aspects, transfection relates to transfection of cell lines or hybrid cell types. In certain aspects, the cell or transfected cell is a cancer cell, a tumor cell, or an immortalized cell. In certain cases, the tumor, cancer, immortalized cell, or cell line is induced, while in other cases, the tumor, cancer, immortalized cell, or cell line naturally enters a respective state or condition. In some aspects, the cell or cell line can be A549, B cell, B16, BHK-21, C2C12, C6, CaCo-2, CAP / , CAP-T, CHO, CHO2, CHO-DG44, CHO-K1, COS-1, Cos-7, CV-1, dendritic cell, DLD-1, embryonic stem cell (ES) cell or derivative, H1299, HEK, 293, 293T, 293FT, Hep G2, hematopoietic stem cells, HOS, Huh-7, induced pluripotent stem (iPS) cells or derivatives, Jurkat, K562, L5278Y, LNCaP, MCF7, MDA-MB-231, MDCK, mesenchymal cells, Min-6, monocytes, Neuro2a, NIH3T3, NIH3T3L1, K562, NK cells, NS0, Panc-1, PC12, PC-3, peripheral blood cells, plasma cells, primary fibroblasts, RBL, murine renal carcinoma cells, RLE, SF21, SF9, SH-SY5Y, SK-MES-1, SK-N-SH, SL3, SW403, stimulus-triggered acquisition of pluripotency (STAP) cells or derivatives SW403, T cells, THP-1, tumor cells, U2OS, U937, peripheral blood lymphocytes, expanded T cells, hematopoietic stem cells or Vero cells.

[0483]

V. Test kit

[0484] Certain aspects of the invention also relate to a kit comprising the disclosed composition or the disclosed composition of the implementation method. In some embodiments, the kit can be used to detect the presence of TdT peptide in a sample. In some embodiments, the kit can be used to detect the presence of TdT peptide in a sample by binding to an antibody to the TdT peptide on the surface of cancer cells. In some embodiments, the kit comprises, comprises at least or comprises at most 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, 50, 100, 500, 1,000 or more probes, primers or primer sets, synthetic molecules or inhibitors, or any value or range and combination derivable therein. In some embodiments, the kit comprises one or more polypeptides capable of binding to the TdT peptide disclosed herein. For example, the kit may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Fabs disclosed herein for detecting TdT peptides. In some embodiments, the kit comprises a detection pair. In some embodiments, the kit comprises an enzyme. In some embodiments, the kit comprises a substrate for the enzyme.

[0485] The kit may comprise components, which may be packaged individually or placed in a container, such as a tube, bottle, vial, syringe or other suitable container means.

[0486] Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided separately at the same concentration as the other components in solution. The component concentration may be 1×, 2×, 5×, 10×, or 20× or higher.

[0487] The kit may comprise any device for obtaining a sample from an individual.

[0488] Kits for use in prognostic or diagnostic applications using the disclosed probes, synthetic nucleic acids, non-synthetic nucleic acids, and / or inhibitors are disclosed as part of the disclosure. In certain aspects, negative and / or positive control nucleic acids, probes, and inhibitors are included in some kit embodiments.

[0489] The kit can also include instructions for use. For example, in some embodiments, the kit includes instructions for detecting cancer in a sample.

[0490] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein, and that different embodiments can be combined. The claims originally filed are intended to encompass claims that rely in various respects on any filed claim or combination of filed claims.

[0491] [VI. Embodiment]

[0492] The following examples are included to demonstrate certain embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques found by the inventors to work in the practice of the present invention. However, it will be appreciated by those skilled in the art that many changes may be made in the disclosed specific embodiment schemes without departing from the spirit and scope of the present disclosure, and still similar or similar results may be obtained.

[0493] [Example 1: TdT-specific cytotoxic receptor and method of use thereof]

[0494] Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in T-cell and B-cell progenitors that catalyzes the transfer of nucleotides during DNA rearrangement (Kunkel et al., 1986; Desiderio et al., 1984). TdT is expressed in up to 95% of T- and B-lineage ALL and is widely used as a diagnostic marker for these malignancies (Drexler et al., 1986). In addition, TdT expression is frequently detected in poorly differentiated AML (Patel et al., 2012; Drexler et al., 1993). Off-target activity of TdT in immature progenitors has been associated with leukemogenesis (Borrow et al., 2019a; Borrow et al., 2019b). The absence of TdT expression in primitive hematopoietic progenitors or mature peripheral lymphocytes makes it an ideal therapeutic target for leukemia. However, the TdT-specific TCR is effectively deleted in the thymus, and the nuclear localization of TdT precludes its targeting by conventional CARs. Therefore, it is thought that chimeric receptors that recognize TdT-derived peptides can be used to target tumor cells expressing TdT in the context of MHC I. A meta-analysis of surface peptides presented by TdT+HLAA2+ leukemia cells was performed and TdT-derived peptides presented in the context of the dominant class I MHC allele HLA-A02 in the Western world were identified (Klatt et al., 2020). Using recombinant HLA-A02:01 / TdT peptide complexes, the inventors screened two independent phage display libraries consisting of >2×10 12The humanized scFv and humanized camel VHH binders were composed of four clones that recognized TdT / HLA-A02:01 but not control peptide / HLA-A02:01 complexes. Since each individual peptide occupies only a very small fraction of the total HLA-A molecules, the surface density of TdT / HLA-A02 complexes is expected to be relatively low and may be below the optimal detection threshold mediated by CAR. To address this possibility, for each binder, a second-generation TdT-specific CAR and TdT-specific chimeric TCR (cTCR) were generated to take advantage of the modular CAR architecture and highly sensitive TCR signaling, which is able to detect only a few cognate peptide / HLA molecules on the cell surface (Purbhoo et al., 2004; Sykuley et al., 1996)( Figure 3 A). In the cTCR construct, the TdT binder is fused to the mouse constant TCR α and / or β chains to achieve specific dimerization and efficient integration with the CD3 signaling complex in human T cells ( Figure 3 A). Most TdT-specific receptors are expressed on the cell surface of primary human T cells following gamma-retroviral transduction, with most binder variants showing high levels of expression in transgenic T cells ( Figure 3 B) and amplification ( Figure 3 C). Expression of TdT-specific CAR in T cells resulted in a moderate reduction in microdifferentiated CD27+CD45RA+ T cells, likely due to predominant CAR signaling, whereas expression of TdT.cTCR did not affect T cell differentiation status ( Figure 3 D). Cytotoxicity of T cells expressing TdT-specific constructs was assessed in co-culture with the TdT+HLA-A02+ leukemia cell line BV173 for 72 hours. One TdT-specific cTCR construct exerted robust cytotoxicity against leukemia cells, whereas other clones showed lower activity ( Figure 3 E). Notably, T cells expressing CARs with the same TdT binder showed only minimal activity, indicating that CAR sensitivity was insufficient to elicit optimal T cell activation.

[0495] To evaluate the specificity of cTCR T cells, malignant and normal lymphocytes were co-cultured for 72 h, and the number of residual target cells was quantified using flow cytometry with bead counting. TdT.cTCR T cells were highly cytotoxic against TdT+ / HLA-A2+BV173 and NALM6 leukemias, but not against negative control CCRF-CEM (TdT+ / HLA-A2-) and THP-1 (TdT- / HLA-A2+) cells ( Figure 4A) and TdT- / HLA-A2+ cell lines GDM-1, Jeko-1, and Loucy had no activity. Next, TdT.cTCR T cells were generated from HLA-A02+ donors and subjected to a 24-hour co-culture assay with autologous T cells and B cells freshly isolated from peripheral blood. No cytotoxicity against normal T and B lymphocytes was observed after co-culture with TdT.cTCR T cells, while control CD19 CAR T cells expectedly killed autologous CD19+ B cells ( Figure 4 B). Expression of TdT.cTCR on HLA-A02+ T cells also did not produce fratricide and did not affect their ex vivo expansion. These data suggest that TdT.cTCR specifically recognizes TdT peptides in the context of HLA-A02 and does not elicit off-target activity against normal TdT-negative lymphocytes.

[0496] Since the TdT-specific binders identified in the phage display screen recognized the TdT / HLA-A02 complex without the help of the CD8 coreceptor, it was assumed that the TdT.cTCR would effectively redirect both CD4+ and CD8+ T cells to fight leukemia. Indeed, both CD4+ and CD8+ T cells showed robust degranulation (as measured by CD107a staining) and production of IFNγ and TNFα ( Figure 5 This activity was abolished by removing surface class I MHC expression using b2M CRISPR knockout, indicating that this activity of CD4+ and CD8+ TdT.cTCR T cells is class I MHC specific ( Figure 5 ). Therefore, unlike conventional tumor antigen-specific TCRs, TdT.cTCRs can engage both the CD4+ and CD8+ T cell arms of the immune response against leukemia.

[0497] The above results indicate that TdT targeting is most effective through activation of conventional TCR signaling. In addition to cTCR transgenic T cells, the TCR pathway can also be engaged using soluble bispecific T cell engagers (BiTEs) in which a tumor antigen-specific binder is fused to an anti-CD3 moiety, thereby inducing TCR crosslinking and T cell activation / degranulation against target cells. To test whether unmodified T cells can be redirected against TdT+ leukemias with BiTEs, TdT-specific engagers were designed and expressed in producer cells. Unmodified untransduced T cells and TdT.CAR and TdT.cTCR T cells were subjected to a standard 72-hour co-culture assay against the TdT+ / HLA-A02+ leukemia cell lines BV173 and NALM-6. While control untransduced T cells had no measurable activity against leukemias in the absence of BiTEs, the addition of soluble BiTEs resulted in robust elimination of both leukemia targets by unmodified T cells, comparable to TdT.cTCR T cells ( Figure 6 Collectively, these results demonstrate that TdT-specific cTCR and BiTE constructs have potent and specific anti-leukemic activity compared with CAR-based modalities and support the feasibility and efficacy of using these agents to target TdT+ leukemias.

[0498] Figure 7 One embodiment of a chimeric TCR is shown, wherein the V H and V L The domains are present on a single TCRα chain, in contrast to the STAR receptors that contain the variable heavy and variable light regions on separate chains of the chimeric TCR (Liu et al., Sci Transl Med. 2021; 13(586):eabb5191). Figure 8 cTCR and STAR expression on T cells after retroviral transduction is shown. Fig. 9 The cytotoxicity of T cells expressing cTCR and STAR against BV173 cell line (TdT+, HLA-A2+) was shown. In particular, the transduced cells were co-cultured with a representative cancer cell line of mixed lineage leukemia (MLL) cells for comparison. In another embodiment, cTCR and BiTE molecules are retrovirally transduced into T cells ( Fig.10 ), Fig.11 Evidence for the cytotoxicity of cTCR T cells and BiTE T cells against a representative cancer cell line of MLL cells (TdT+, HLA-A2+) is provided.

[0499] ***

[0500] All methods disclosed and claimed herein can be made and performed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the methods and steps of the methods or the order of steps described herein without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain chemically and physiologically related reagents may be substituted for the reagents described herein while the same or similar results will be obtained. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be in accordance with the spirit, scope, and concept of the invention as defined by the appended claims.

[0501]

References

[0502] The following references, to the extent they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0503] Borrow, J., Dyer, SA, Akiki, S. & Griffiths, MJTerminaldeoxynucleotidyltransferase promotes acute myeloid leukemia by primingF LT3-ITD replicationslippage. Blood 134, 2281-2290 (2019a).

[0504] Borrow, J., Dyer, SA, Akiki, S. & Griffiths, MJ Molecularroulette: nucleophosmin mutations in AML are orchestrated through N-nucleotide addition by TdT. Blood 134, 2291-2303 (2019b).

[0505] Desiderio,SVet al.Insertion of N regions into heavy-chain genesiscorrelated with expression of terminal deoxytransferase in B cells.Nature311,752-755(1984).

[0506] Drexler,H.G.,Menon,M.&Minowada,J.Incidence of TdTpositivity in casesof leukemia and lymphoma.Acta Haematol 75,12-17(1986).

[0507] Drexler,H.G.,Sperling,C.&Ludwig,W.D.Terminaldeoxynucleotidyltransferase(TdT)expression in acute myeloid leukemia.Leukemia 7,1142-1150(1993).

[0508] Gudipati,V.et al.Inefficient CAR-proximal signaling bluntsantigensensitivity.Nature Immunology 21,848-856(2020).

[0509] Klatt,M.G.et al.Solving an MHC allele-specific bias in thereportedimmunopeptidome.JCI Insight 5,(2020).

[0510] Kunkel,T.A.,Gopinathan,K.P.,Dube,D.K.,Snow,E.T.&Loeb,L.A.Rearrangements of DNA mediated by terminal transferase.Proc NatlAcad SciU S A 83,1867-1871(1986).

[0511] Patel,J.L.et al.The immunophenotype of T-lymphoblastic lymphomainchildren and adolescents:a Children's Oncology Group report.Br JHaematol 159,454-461(2012).

[0512] Purbhoo,M.A.,Irvine,D.J.,Huppa,J.B.&Davis,M.M.T cellkilling does notrequire the formation of a stable mature immunologicalsynapse.Nat Immunol 5,524-530(2004).

[0513] Sykulev,Y.,Joo,M.,Vturina,I.&Eisen,H.N.Evidence that asingle peptide-MHC complex on a target cell can elicit a cytolytic T cellresponse.Immunity4,565-571(1996).

[0514] Watanabe,K.et al.Target antigen density governs the efficacy ofanti-CD20-CD28-CD3ζchimeric antigen receptor-modified effector CD8+T cells.JImmunol 194,911-920(2015).

Claims

1. An antibody or antigen-binding fragment comprising any one or more of SEQ ID NOs: 4-9 or 22-27.

2. An antibody or antigen-binding fragment comprising a heavy chain variable region having at least 80% sequence identity to SEQ ID NOs: 22-24.

3. The antibody or antigen-binding fragment according to claim 2, wherein: The antibody or antigen-binding fragment further comprises a light chain variable region having at least 80% sequence identity to SEQ ID NOs: 25-27.

4. An antibody or antigen-binding fragment comprising a light chain variable region having at least 80% sequence identity to SEQ ID NOs: 25-27.

5. The antibody or antigen-binding fragment according to claim 4, wherein The antibody or antigen-binding fragment comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NOs: 22-24.

6. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 28-30, respectively, and Wherein, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having at least 80% sequence identity with SEQ ID NOs: 31 to 33, respectively.

7. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 28 to 30, respectively, and Wherein, the light chain variable regions respectively comprise LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 31, 33 or "DVS".

8. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 34-36, respectively, and Wherein, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having at least 80% sequence identity with SEQ ID NO: 37, 39 or "DAS", respectively.

9. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 34 to 36, respectively, and Wherein, the light chain variable regions respectively comprise LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 37, 39 or "DAS".

10. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 40-42, respectively, and Wherein, the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having at least 80% sequence identity with SEQ ID NO: 43, 45 or "DDN", respectively.

11. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NOs: 40 to 42, respectively, and Wherein, the light chain variable regions respectively comprise LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 43, 45 or "DDN".

12. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:22, and wherein the light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

25.

13. The antibody or antigen-binding fragment according to claim 12, wherein: The heavy chain variable region comprises the sequence of SEQ ID NO:

22.

14. The antibody or antigen-binding fragment according to claim 12 or 13, wherein The light chain variable region comprises the sequence of SEQ ID NO:

25.

15. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:23, and wherein the light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

26.

16. The antibody or antigen-binding fragment according to claim 12, wherein: The heavy chain variable region comprises the sequence of SEQ ID NO:

23.

17. The antibody or antigen-binding fragment according to claim 12 or 13, wherein The light chain variable region comprises the sequence of SEQ ID NO:

26.

18. An antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, in, The heavy chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:24, and wherein the light chain variable region comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:

27.

19. The antibody or antigen-binding fragment according to claim 12, wherein: The heavy chain variable region comprises the sequence of SEQ ID NO:

24.

20. The antibody or antigen-binding fragment according to claim 12 or 13, wherein The light chain variable region comprises the sequence of SEQ ID NO:

27.

21. The antibody or antigen-binding fragment according to any one of claims 1 to 20, wherein The antibody or antigen-binding fragment is about 10 -3 M~about 10 -9 M of K D Binds to TdT peptide.

22. The antibody or antigen-binding fragment according to any one of claims 1 to 21, wherein The antibody is: a human antibody, a humanized antibody, a recombinant antibody, a chimeric antibody, an antibody derivative, a neutralizing antibody, a modified antibody, a bispecific antibody, a monoclonal antibody, a single domain antibody or a single chain antibody.

23. The antigen-binding fragment according to any one of claims 1 to 22, wherein The antigen binding fragment is a single chain variable fragment (scFv), F(ab')2, Fab', Fab, Fv or rIgG.

24. The antibody or antigen-binding fragment of any one of claims 1 to 23, further defined as being comprised in a chimeric polypeptide.

25. The antibody or antigen-binding fragment according to claim 24, wherein The chimeric polypeptide is a receptor or a multispecific antibody.

26. A polypeptide comprising the antigen-binding fragment of any one of claims 1 to 25.

27. The polypeptide according to claim 26, wherein The polypeptide comprises at least two antigen-binding fragments, wherein each antigen-binding fragment is independently selected from the antigen-binding fragment of any one of claims 1 to 25.

28. The polypeptide according to claim 26 or 27, wherein The polypeptide is multivalent.

29. The polypeptide according to any one of claims 26 to 28, wherein The polypeptides are bispecific.

30. The polypeptide according to claim 29, wherein The polypeptide is a bispecific antibody, wherein The first antigen binding domain of the antibody binds to the TdT peptide, and The second antigen binding domain of the antibody binds CD3.

31. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 25.

32. The composition according to claim 31, wherein The composition comprises a chimeric receptor or a multispecific antibody.

33. The composition according to claim 32, wherein The chimeric receptor is a chimeric antigen receptor or a chimeric T cell receptor (TCR) comprising a TCR alpha chain and a beta chain.

34. The composition according to claim 32, wherein The multispecific antibody is a bispecific antibody.

35. The composition according to claim 32 or 34, wherein The composition is a bispecific T cell engager.

36. The composition according to any one of claims 31 to 35, wherein The composition comprises a pharmaceutical excipient.

37. The composition according to any one of claims 31 to 36, wherein The composition also comprises an adjuvant.

38. The composition according to any one of claims 31 to 37, wherein The compositions are formulated for administration parenterally, in situ, intradermally, subcutaneously, orally, transdermally, intramuscularly, intraperitoneally, intraorbitally, implanted, inhaled, intracerebroventricularly, intranasally, intraarterially, intratracheally, intrapleurally, intratumorally, endoscopically, intralesionally, intracranially, transdermally, topically, systemically, by perfusion, in a tumor microenvironment, and / or by intravenous injection.

39. The composition according to any one of claims 31 to 38, wherein The composition comprises at least two antibodies or antigen-binding fragments.

40. One or more nucleic acids encoding the antibody or antigen-binding fragment of any one of claims 1 to 25 or the polypeptide of any one of claims 26 to 30.

41. A nucleic acid encoding a TdT peptide-specific antibody, wherein: The nucleic acid has at least 70% sequence identity to one of SEQ ID NOs: 4, 6 or 8.

42. A vector comprising the nucleic acid of claim 41.

43. A host cell comprising the nucleic acid of claim 41 or the vector of claim 42.

44. The host cell according to claim 43, wherein The host cell is a human cell, an immune cell, an immune effector cell, a B cell, a T cell, a Chinese hamster ovary cell, a NS0 mouse myeloma cell or a PER.C6 cell.

45. A method for preparing a cell, comprising transferring the nucleic acid of claim 41 or the vector of claim 42 into the cell.

46. ​​The method of claim 45, wherein: The method further comprises culturing the cell under conditions permitting expression of the polypeptide from the nucleic acid.

47. The method of claim 46, wherein: The method further comprises isolating the expressed polypeptide.

48. A method according to any one of claims 45 to 47, wherein: The cells are human cells, B cells, T cells, Chinese hamster ovary cells, NS0 mouse myeloma cells or PER.C6 cells.

49. A method for producing a polypeptide, comprising transferring the nucleic acid of claim 41 or the vector of claim 42 into a cell and isolating the polypeptide expressed from the nucleic acid.

50. The method of claim 49, wherein: The cells are: human cells, B cells, T cells, Chinese hamster ovary cells, NS0 mouse myeloma cells or PER.C6 cells.

51. A method for treating or preventing cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of The antibody or antigen-binding fragment according to any one of claims 1 to 25, The polypeptide according to any one of claims 26 to 30, A composition according to any one of claims 31 to 39, or A host cell according to claim 43 or 44.

52. The method of claim 51, wherein: The subject is a human subject.

53. The method according to claim 51 or 52, wherein: The cancer is a blood cancer.

54. The method of claim 51 or 52, wherein: The cancer is a solid tumor.

55. The method according to any one of claims 51 to 54, wherein: The subject has one or more symptoms of cancer.

56. The method according to claims 51 to 54, wherein: The subject does not have any symptoms of cancer.

57. The method according to any one of claims 51 to 56, wherein: The subject has been diagnosed with cancer.

58. The method according to any one of claims 51 to 56, wherein: The subject has not been diagnosed with cancer.

59. The method according to any one of claims 51 to 58, wherein: The subject has not been previously vaccinated with a cancer vaccine.

60. The method according to any one of claims 51 to 59, wherein: The antibodies, antigen-binding fragments, polypeptides or cells are administered parenterally, in situ, intradermally, subcutaneously, orally, transdermally, intramuscularly, intraperitoneally, intraorbitally, implanted, inhaled, intracerebroventricularly, intranasally, intraarterially, intratracheally, intrapleurally, intratumorally, endoscopically, intralesionally, intracranially, transdermally, topically, systemically, by perfusion, in a tumor microenvironment and / or by intravenous injection.

61. The method according to any one of claims 51 to 60, wherein: The subject has previously been treated for cancer.

62. The method according to any one of claims 51 to 61, wherein: The subject is administered additional cancer therapy or drug therapy.

63. The method of claim 62, wherein: The additional cancer treatment or drug therapy includes surgery, radiation therapy, chemotherapy, drug therapy, hormone therapy, or a combination thereof.

64. A method for reducing the risk of cancer, preventing cancer or delaying the onset of cancer in an individual, comprising administering to the subject a therapeutically effective amount of The antibody or antigen-binding fragment according to any one of claims 1 to 25, The polypeptide according to any one of claims 26 to 30, A composition according to any one of claims 31 to 39, or A host cell according to claim 43 or 44.

65. The method of claim 64, wherein: The individual has one or more risk factors for cancer.

66. The method of claim 65 or 64, wherein the individual is a smoker, is over 50 years of age, has a genetic marker, has a personal history, has a family history, has sun damaged skin, or a combination thereof.

67. A method of evaluating a sample from a subject, the method comprising contacting a biological sample from the subject, or an extract thereof, with at least one of: The antibody or antigen-binding fragment according to any one of claims 1 to 25, The polypeptide according to any one of claims 26 to 30, A composition according to any one of claims 31 to 39, or A host cell according to claim 43 or 44.

68. The method of claim 67, wherein: The at least one antibody, antigen-binding fragment or polypeptide is operably linked to a detectable label.

69. The method of claim 67 or 68, wherein: The method further comprises incubating the antibody, antigen-binding fragment or polypeptide under conditions that allow the antibody, antigen-binding fragment or polypeptide to bind to the antigen in the biological sample or an extract thereof.

70. The method according to any one of claims 67 to 69, wherein: The method further comprises detecting binding of the antigen to the antibody, antigen binding fragment or polypeptide.

71. The method according to any one of claims 67 to 70, wherein: The method further comprises contacting the biological sample with at least one capture antibody, antigen, or polypeptide.

72. The method of claim 71, wherein: The at least one capture antibody, antigen-binding fragment or polypeptide comprises at least one antibody according to claims 1-15.

73. The method of claim 71 or 72, wherein: The capture antibody is attached to a solid support.

74. The method according to any one of claims 67 to 73, wherein: The biological sample includes a blood sample, a urine sample, a stool sample, a nasopharyngeal sample, a cerebrospinal fluid sample, a cheek scrape sample, a nipple aspiration sample, a biopsy sample, or a combination thereof.

75. The method of any one of claims 67 to 74, wherein the individual has a higher risk of cancer than the general population.

76. A method for diagnosing cancer in a subject, the method comprising contacting a biological sample from the subject, or an extract thereof, with at least one antibody, antigen-binding fragment of any one of claims 1 to 25, or with a polypeptide of any one of claims 26 to 30.

77. The method of claim 76, wherein: The at least one antibody, antigen-binding fragment or polypeptide is operably linked to a detectable label.

78. The method of claim 76 or 77, wherein: The method further comprises incubating the antibody, antigen-binding fragment or polypeptide under conditions that allow the antibody, antigen-binding fragment or polypeptide to bind to the antigen in the biological sample or an extract thereof.

79. The method according to any one of claims 76 to 78, wherein The method further comprises detecting binding of an antigen to the antibody, antigen binding fragment or polypeptide.

80. The method according to any one of claims 76 to 79, wherein: The method further comprises contacting the biological sample with at least one capture antibody, antigen, or polypeptide.

81. The method of claim 80, wherein: The at least one capture antibody, antigen or polypeptide comprises at least one antibody, antigen or polypeptide according to claims 1 to 15.

82. The method of claim 80 or 81, wherein the capture antibody is attached to a solid support.

83. The method according to any one of claims 76 to 82, wherein: The biological sample includes a blood sample, a urine sample, a stool sample, a nasopharyngeal sample, a cerebrospinal fluid sample, a cheek scrape sample, a nipple aspiration sample, a biopsy sample, or a combination thereof.

84. A method of treating cancer in an individual comprising providing to the individual determined to have cancer cells expressing a TdT peptide a therapeutically effective amount of The antibody or antigen-binding fragment according to any one of claims 1 to 25, The polypeptide according to any one of claims 26 to 30, A composition according to any one of claims 31 to 39, or The host cell according to claim 43 or 44, The assay uses: The antibody or antigen-binding fragment according to any one of claims 1 to 25, The polypeptide according to any one of claims 26 to 30, A composition according to any one of claims 31 to 39, or A host cell according to claim 43 or 44.

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