Human CD6-binding molecule

Abstract

This specification provides human differentiation antigen group 6 (CD6) binding molecules and nucleic acid sequences encoding such molecules. In certain embodiments, this specification provides a first single-monomer variable antibody domain (SMVAD) comprising a specific CDR and, optionally, a human CD6 binding molecule (e.g., a nanobody) having a second single-monomer variable antibody domain, and a method for treating T-cell-related diseases (e.g., cancer, e.g., T-cell lymphoma) using such molecules. In certain embodiments, the SMVAD comprises a camelid, human, or humanized framework region.

Description

Detailed Description of the Invention

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[0001] [Technical Field] This application claims priority to U.S. Provisional Application No. 63 / 454,127, filed Mar. 23, 2023, which is hereby incorporated by reference in its entirety.

[0002] This invention was made with government support under grant EY025373 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] [Sequence Listing]<0000​​​​​​​​​​​​T-cell lymphoma is a rare form of cancerous lymphoma that affects T cells. Lymphomas primarily arise from the uncontrolled proliferation of T cells and can become cancerous. T-cell lymphoma is classified as non-Hodgkin lymphoma (NHL) and accounts for less than 15% of all non-Hodgkin diseases in this category. T-cell lymphoma is often classified as intermediate-grade (rapidly growing) or low-grade (slowly growing) based on its growth pattern. The cause of T-cell lymphoma is not definitively established, but it is associated with various risk factors and viruses (e.g., Epstein-Barr virus (EBV) and human T-cell leukemia virus 1 (HTLV-1)).

[0006] The prognosis and treatment of T-cell lymphoma can vary significantly depending on the specific type of lymphoma and its growth pattern. Due to its rarity and large variability among different subtypes, the prognosis for T-cell lymphoma is significantly worse than that of other non-Hodgkin lymphomas. Treatment for T-cell lymphoma is often similar to that for other non-Hodgkin lymphomas, with initial treatment consisting of chemotherapy and / or radiotherapy. The effectiveness of these treatments often varies among subtypes, and most result in inadequate outcomes with high relapse rates.

[0007] [Overview of the prefecture] This specification provides human differentiation antigen group 6 (CD6) binding molecules and nucleic acid sequences encoding such molecules. In certain embodiments, this specification provides a first single-monomer variable antibody domain (SMVAD) comprising a specific CDR and, optionally, a human CD6 binding molecule (e.g., a nanobody) having a second single-monomer variable antibody domain, and a method for treating T-cell-related diseases (e.g., cancer, e.g., T-cell lymphoma) using such molecules. In certain embodiments, the SMVAD comprises a camelid, human, or humanized framework region. In some embodiments, the human CD6 binding molecule is a human CD6 binding molecule.

[0008] In some embodiments, this specification provides compositions comprising a human differentiation antigen group 6 (CD6) binding molecule, or one or more nucleic acid molecules encoding the human CD6 binding molecule, wherein the human CD6 binding molecule comprises a first single monomer variable antibody domain (SMVAD) comprising: A) SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78; or a CDR1 amino acid sequence comprising SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78 having one or two conservative amino acid changes; B) SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 3 C) CDR2 amino acid sequences including 1, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79; or C) CDR2 amino acid sequences including SEQ ID NOs. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79 having one or two conservative amino acid changes, and C) SEQ ID NOs. 4, 8, 1 CDR3 amino acid sequences containing SEQ ID NOs: 2, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80; or CDR3 amino acid sequences containing SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80 having one or two conservative amino acid changes.

[0009] In certain embodiments, methods for treating or preventing T-cell-related diseases or conditions are provided herein, the methods comprising treating a subject with a composition comprising a human differentiation antigen group 6 (CD6) binding molecule, or an expression vector comprising one or more nucleic acid molecules encoding the CD6 binding molecule, the subject having or suspecting to develop a T-cell-related disease or condition. In some embodiments, the T-cell-related disease includes cancer, optionally, the cancer includes T-cell lymphoma. In other embodiments, the T-cell-related disease includes acute respiratory distress syndrome (ARDS), cytokine release syndrome of a Covid-19 subject, or acute graft-versus-host disease (aCGDH). In further embodiments, the T-cell-related disease includes lupus nephritis, uncontrolled asthma, psoriasis, or multiple sclerosis. In certain embodiments, the human CD6 binding molecule is conjugated to a cytotoxic agent, optionally, the cytotoxic agent includes monomethyl auristatin (MMAE).

[0010] In other embodiments, methods for detecting human differentiation antigen group 6 (CD6) in a sample are provided herein, the methods comprising: a) contacting the sample with a human CD6-binding molecule described above and herein (if the sample is suspected to contain human CD6 and the human CD6-binding molecule is present in the sample, it will form a complex with human CD6); and b) detecting the presence or absence of the complex in the sample. In some embodiments, the sample is from a subject suffering from or suspected to develop a T cell-related disease or condition. In other embodiments, the human CD6-binding molecule includes a detectable label. In further embodiments, the method further comprises contacting the sample with a conjugate molecule capable of binding to the human CD6-binding molecule, the conjugate molecule including a detectable label.

[0011] In certain embodiments, the first SMVAD further includes four framework regions, the four framework regions being camelid, humanized, or human framework regions. In further embodiments, the human CD6-binding molecule further comprises a second SMVAD including: D) SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78; or a CDR1 amino acid sequence including SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78 having one or two conservative amino acid changes; E) SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 7 9; or a CDR2 amino acid sequence containing SEQ ID NOs. 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79 having one or two conservative amino acid changes, and F) SEQ ID NOs. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80 having one or two conservative amino acid changes.

[0012] In further embodiments, the human CD6-binding molecule further includes a linker bound to both the first SMVAD and the second SMVAD. In other embodiments, one or more nucleic acid molecules include i) a first nucleic acid sequence encoding the first SMVAD and optionally further encoding a CH2 heavy chain constant region (e.g., human or humanized) and / or a CH3 heavy chain constant region (e.g., human or humanized), and ii) a second nucleic acid sequence encoding the second SMVAD and optionally further encoding a CH2 heavy chain constant region (e.g., human or humanized) and / or a CH3 heavy chain constant region (e.g., human or humanized). In some embodiments, the first SMVAD includes the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, or 77, or the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, or 77, having one, two, three, or four deletions and / or conservative amino acid changes at one or both ends. In other embodiments, the human CD6-binding molecule further includes a CH2 heavy chain constant region and / or a CH3 heavy chain constant region. In some embodiments, the CH2 and / or CH3 heavy chain constant regions are camelid, humanized, or human. In further embodiments, the human CD6-binding molecule includes at least an antigen-binding moiety of a clone 2G1 CD6 nanobody.

[0013] In some embodiments, the compositions, kits, and systems herein further include physiologically acceptable buffers. In certain embodiments, the compositions herein include one or more nucleic acid molecules (e.g., first and second nucleic acid molecules), and optionally, the compositions further include an expression vector, wherein one or more nucleic acid sequences are present within the expression vector. In certain embodiments, the compositions include a human CD6-binding molecule. In some embodiments, the CDR1 amino acid sequence includes SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78; the CDR2 amino acid sequence includes SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79; and the CDR3 amino acid sequence includes SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80.

[0014] [Brief description of the drawing] [Figure 1] The amino acid sequence (SEQ ID NO: 1) of the clone 2G1 CD6 nanobody VHH sequence, including CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3), and CDR3 (SEQ ID NO: 4), is shown.

[0015] [Figure 2] An exemplary step-by-step procedure used to generate CD6 nanobodies is shown.

[0016] [Figure 3] This shows that purified CD6 clone 2G1 binds to human CD6 with similar affinity and is detectable at 0.1 pM.

[0017] [Figure 4] Demonstrates that clone 2G1 nanobodies bind to the T cell line (CD6+) in a dose-dependent manner.

[0018] [Figure 5] An exemplary design of a novel divalent anti-CD6 nanobody based on next-generation CD6-ADC is shown.

[0019] [Figure 6A] This shows that the designed CD6 nanobody selectively binds to CD6 on T cells.

[0020] [Figure 6B] This shows that the designed CD6 nanobody selectively binds to CD6 on T cells.

[0021] [Figure 7] This shows that divalent CD6 nanobodies are internalized in T cells (HuT78).

[0022] [Figure 8] This shows that next-generation CD6-ADCs kill T-cell lymphoma cells more effectively than first-generation CD6-ADCs.

[0023] [Figure 9] This shows that MMAE (monomethyl auristatin) bound to the CD6 nanobody dimer (2G1-2G1) inhibits the proliferation of human T cells (HH cell line).

[0024] [Figure 10] A shows the amino acid sequence (SEQ ID NO: 5) of the clone A1CD601 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 6), CDR2 (SEQ ID NO: 7), and CDR3 (SEQ ID NO: 8). B shows the amino acid sequence (SEQ ID NO: 9) of the clone CD601HH1 nanobody VHH sequence containing CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12). C shows the amino acid sequence (SEQ ID NO: 13) of the clone CD601HH2 nanobody VHH sequence containing CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 16). D shows the amino acid sequence (SEQ ID NO: 17) of the clone CD601HH3 nanobody VHH sequence containing CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19), and CDR3 (SEQ ID NO: 20).

[0025] [Figure 11] A shows the amino acid sequence (SEQ ID NO: 21) of the clone CD601HH4 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO: 24). B shows the amino acid sequence (SEQ ID NO: 25) of the clone CD601HH5 nanobody VHH sequence containing CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO: 27), and CDR3 (SEQ ID NO: 28). C shows the amino acid sequence (SEQ ID NO: 29) of the clone CD601HH6 nanobody VHH sequence containing CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 32). D shows the amino acid sequence (SEQ ID NO: 33) of the clone CD601HH7 nanobody VHH sequence containing CDR1 (SEQ ID NO: 34), CDR2 (SEQ ID NO: 35), and CDR3 (SEQ ID NO: 36).

[0026] [Figure 12] A shows the amino acid sequence (SEQ ID NO: 37) of the clone CD601HH8 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 38), CDR2 (SEQ ID NO: 39), and CDR3 (SEQ ID NO: 40). B shows the amino acid sequence (SEQ ID NO: 41) of the clone CD601HH9 nanobody VHH sequence containing CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43), and CDR3 (SEQ ID NO: 44). C shows the amino acid sequence (SEQ ID NO: 45) of the clone CD601HH10 nanobody VHH sequence containing CDR1 (SEQ ID NO: 46), CDR2 (SEQ ID NO: 47), and CDR3 (SEQ ID NO: 48). D shows the amino acid sequence (SEQ ID NO: 49) of the clone CD601HH11 nanobody VHH sequence containing CDR1 (SEQ ID NO: 50), CDR2 (SEQ ID NO: 51), and CDR3 (SEQ ID NO: 52).

[0027] [Figure 13] A shows the amino acid sequence (SEQ ID NO: 53) of the cloned CD601HH12 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 54), CDR2 (SEQ ID NO: 55), and CDR3 (SEQ ID NO: 56). B shows the amino acid sequence (SEQ ID NO: 57) of the cloned CD601HH13 nanobody VHH sequence containing CDR1 (SEQ ID NO: 58), CDR2 (SEQ ID NO: 59), and CDR3 (SEQ ID NO: 60). C shows the amino acid sequence (SEQ ID NO: 61) of the cloned CD601HH14 nanobody VHH sequence containing CDR1 (SEQ ID NO: 62), CDR2 (SEQ ID NO: 63), and CDR3 (SEQ ID NO: 64). D shows the amino acid sequence (SEQ ID NO: 65) of the cloned CD601HH15 nanobody VHH sequence containing CDR1 (SEQ ID NO: 66), CDR2 (SEQ ID NO: 67), and CDR3 (SEQ ID NO: 68).

[0028] [Figure 14] A shows the amino acid sequence (SEQ ID NO: 69) of the cloned CD601HH16 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 70), CDR2 (SEQ ID NO: 71), and CDR3 (SEQ ID NO: 72). B shows the amino acid sequence (SEQ ID NO: 73) of the cloned CD601HH17 nanobody VHH sequence containing CDR1 (SEQ ID NO: 74), CDR2 (SEQ ID NO: 75), and CDR3 (SEQ ID NO: 76). C shows the amino acid sequence (SEQ ID NO: 77) of the cloned CD601HH18 nanobody VHH sequence containing CDR1 (SEQ ID NO: 78), CDR2 (SEQ ID NO: 79), and CDR3 (SEQ ID NO: 80).

[0029] [Figure 15] Shows the results of a CD6-ADC assay based on the killing of T cell lymphoma cells by a humanized CD6 nanobody. One of the humanized CD6 nanobody clones (HH4) was conjugated with MMAE to develop a next-generation CD6-ADC. T cell lymphoma cell line HH cells were cultured for 72 hours with the new CD6-ADC (HH14-MMAE) or control (hIgG-MMAE) at 0 - 128 nM, and cell killing was quantified using trypan blue to distinguish dead cells from live cells.

[0030] [Definitions] To facilitate understanding of this invention, several terms are defined below.

[0031] As used herein, “nanobody,” “single variable domain” (“VHH”), or “single monomer variable antibody domain” (“SMVAD”) refers to the smallest antigen-binding fragment derived from naturally occurring heavy-chain antibodies, and is known to those skilled in the art. Such nanobodies may originate from antibodies produced in camelid species (e.g., camels, llamas, dromedary camels, alpacas, and guanacos). Nanobodies can also be synthetically produced, for example, by overexpression in bacteria. A single-domain antibody is an antibody in which the complementarity-determining region (CDR) is part of a single-domain polypeptide. Examples include, but are not limited to, heavy-chain antibodies, antibodies naturally lacking a light chain, single-domain antibodies derived from conventional four-chain antibodies, modified antibodies, and single-domain scaffolds other than those derived from antibodies.

[0032] As used herein, the terms “subject” and “patient” refer to any animal (e.g., mammals, e.g., dogs, cats, birds, livestock, preferably humans).

[0033] As used herein, the terms “codon” or “triplet” refer to a group of three adjacent nucleotides that designate one of the naturally occurring amino acids found in a polypeptide. The terms also include codons that do not designate any amino acid. It should also be noted that, due to the degeneracy of the genetic code, there may be many codons that code for the same amino acid. Therefore, many of the bases in the nucleic acid sequences of the present invention can be modified without changing the actual amino acid sequence they encode. This disclosure is intended to encompass all such nucleic acid sequences.

[0034] As used herein, the terms “oligonucleotide having a polypeptide-coding nucleotide sequence,” “polynucleotide having a polypeptide-coding nucleotide sequence,” and “nucleic acid sequence having a peptide-coding nucleotide sequence” mean a nucleic acid sequence containing the coding region of a particular polypeptide. The coding region may exist in the form of, for example, cDNA, genomic DNA, or RNA. If present in the form of DNA, the oligonucleotide or polynucleotide may be single-stranded (i.e., sense strand) or double-stranded. Suitable regulatory elements (e.g., enhancer / promoter, splice junction, polyadenylation signal, etc.) may be located near the coding region of the gene if necessary to enable proper transcription initiation and / or correct processing of primary RNA transcription. Alternatively, the coding region utilized in the expression vector of the present invention may contain endogenous enhancer / promoter, splice junction, intervening sequence, polyadenylation signal, etc., or a combination of both endogenous and exogenous regulatory elements.

[0035] When used in relation to nucleic acids, the term "isolated" typically refers to a nucleic acid sequence that is identified and separated from at least one associated contaminating nucleic acid (e.g., a host cell protein), such as "isolated oligonucleotide," "isolated polynucleotide," or "isolated nucleic acid sequence encoding a differentiation antigen group 6 binding molecule."

[0036] As used herein, the terms “purified” or “purify” refer to the removal of contaminants from a sample. For example, differentiated antigen group 6 binding molecules may be purified by removing contaminating non-immunoglobulin proteins, or by removing immunoglobulins that do not bind to the same antigen. Removal of non-immunoglobulin proteins and / or immunoglobulins that do not bind to a particular antigen increases the proportion of antigen-specific immunoglobulins in the sample. In another example, recombinant antigen-specific polypeptides are expressed in bacterial host cells, and the polypeptides are purified by removing host cell proteins, thereby increasing the proportion of recombinant antigen-specific polypeptides in the sample.

[0037] [Modes for carrying out the invention] This specification provides human differentiation antigen group 6 (CD6) binding molecules and nucleic acid sequences encoding such molecules. In certain embodiments, this specification provides a first single-monomer variable antibody domain (SMVAD) (also known as a "nanobody") comprising a specific CDR, and optionally a human CD6 binding molecule (e.g., a nanobody) having a second single-monomer variable antibody domain, and a method for treating T-cell-related diseases (e.g., cancer, e.g., T-cell lymphoma) using such molecules. In certain embodiments, the SMVAD comprises a camelid, human, or humanized framework region.

[0038] In certain embodiments, the nanobodies (SMVADs) according to this disclosure generally comprise a single amino acid chain (preferably the sequence FR1-CDR1-FR2-CDR2-FR3-(optionally, CDR3)-FR4) which is thought to include four “framework sequences” or FRs and two or three “complementarity-determining regions” or CDRs. Non-limiting examples of nanobodies of this disclosure are described in further detail herein. It should be apparent that the framework regions of the nanobodies may also contribute to antigen binding. However, it should be noted that parts, fragments, analogs, or derivatives of nanobodies (as further described herein) are not limited in particular, in terms of their length and / or size, as long as such parts, fragments, analogs, or derivatives satisfy the further requirements outlined herein and are preferably suitable for the purposes described herein.

[0039] The terms “nanobody” and “SMVAD” are not limited in their broadest sense to a specific biological source or specific preparation method. For example, the nanobody of this disclosure can generally be obtained by: (1) isolation of the VHH domain of a naturally occurring heavy chain antibody; (2) expression of a nucleotide sequence encoding a naturally occurring VHH domain; or (3) “humanization” of a naturally occurring VHH domain, or expression of a nucleic acid encoding such a humanized VHH domain (see, for example: Sulea, Humanization of Camelid Single Domain Antibodies, Methods Mol Biol. 2022;2446:299-312 and Vincke et al., General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold, The J.of Bio.Chem. Vol.284, No.5, pp.3273-3284, January). 30,2009 (both of these are incorporated herein in whole, particularly with respect to methods for humanizing nanobodies); (4) “Camelization” of a naturally occurring VH domain from any animal species (in particular mammalian species (e.g., human)), or expression of nucleic acids encoding such a camelized VH domain; (5) “Camelization” of a “domain antibody” or “Dab” described in the Art, or expression of nucleic acids encoding such a camelized VH domain; (6) Use of synthetic or semi-synthetic techniques to prepare proteins, polypeptides, or other amino acid sequences known by themselves; (7) Preparation of nucleic acids encoding nanobodies using techniques for nucleic acid synthesis known by themselves, followed by expression of the nucleic acids thus obtained; and / or (8) Any combination of one or more of the above.

[0040] Due to their small size and unique biophysical properties, nanobodies generally outperform conventional antibody fragments in recognizing rare or hidden epitopes and binding to cavities or active sites of protein targets. Furthermore, the nanobodies described herein can be designed as bispecific and bivalent antibodies, or conjugated to reporter molecules. Nanobodies are generally easy to fabricate and are stable, rigid single-domain proteins capable of surviving in the gastrointestinal system.

[0041] Studies conducted during the development of the embodiments described herein led to the development of VHH sequences for a chimeric antibody (A1CD601 VHH) and 18 humanized antibodies (CD601HH1-18 VHH). The VHH sequences are shown in Figures 10-14, and their alignments are shown in Figure 15. The characteristics of four of these antibodies (CD601HH8, 10, 12, and 14HuG1Fc) and the chimeric antibody (A1CD601 HuG1Fc) are shown in Table 1 below. The sequences (SEQ ID NO: 83) used for HuG1Fc expression in these antibodies are shown in Table 2 below.

[0042] [Table 1]

[0043] [Table 2]

[0044] [Table 3]

[0045] Other constant regions known in the art, other than those listed in Table 3, may also be used in conjunction with the VHHs of this specification, but these are merely examples.

[0046] The amino acid residues of the nanobodies are generally numbered according to the general numbering for VH domains given by Kabat et al., as applied to the VHH domains derived from camelid animals in the following paper: Riechmann and Muyldermans, J Immunol Methods 1999 Dec 10;231(1-2):25-38 (incorporated herein by reference). According to this numbering, nanobodies FR1 contains amino acid residues at positions 1-30, nanobodies CDR1 contains amino acid residues at positions 31-35, nanobodies FR2 contains amino acids at positions 36-49, nanobodies CDR2 contains amino acid residues at positions 50-65, nanobodies FR3 contains amino acid residues at positions 66-94, nanobodies CDR3 contains amino acid residues at positions 95-102, and nanobodies FR4 contains amino acid residues at positions 103-113. It should be noted that, with respect to the VH domain and VHH domain, it is well known in the art that the total number of amino acid residues within each CDR may vary and may not match the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions indicated by Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than permitted by Kabat numbering). This generally means that Kabat numbering may or may not match the actual numbering of amino acid residues in the actual sequence. However, generally speaking, according to Kabat numbering, regardless of the number of amino acid residues within the CDR, position 1 in Kabat numbering corresponds to the start of FR1 and vice versa; position 36 in Kabat numbering corresponds to the start of FR2 and vice versa; position 66 in Kabat numbering corresponds to the start of FR3 and vice versa; and position 103 in Kabat numbering corresponds to the start of FR4 and vice versa.

[0047] Nanobodies possess numerous unique structural and functional properties, which make isolated SMVADs and proteins containing them highly advantageous for use as functional antigen-binding domains or proteins. In particular, SMVADs "designed" to functionally bind to antigens that lack a light chain variable domain, and to antigens with which they have no interaction, can function as single, relatively small, functional antigen-binding structural units, domains, or proteins. This distinguishes nanobodies from the VH and VL domains of conventional four-chain antibodies. These are generally not suitable for practical use as single antigen-binding proteins or domains on their own, but need to be combined in various forms to provide functional antigen-binding units (e.g., conventional antibody fragments (e.g., Fab fragments); such as ScFv fragments consisting of a VH domain covalently bonded to a VL domain).

[0048] In certain embodiments, the SMVAD (nanobody) can be further modified with one or more other amino substitutions while maintaining its activity as a CD6-binding molecule. In certain embodiments, the substitutions are made in the framework region rather than the CDR domain. For example, amino acid substitutions can be made at one or more positions where the substitution is for similarly hydrophilic amino acids. The importance of the hydroxyl amino acid index in conferring interactive biological function to proteins is generally understood in the art. The relative hydroxyl properties of amino acids contribute to the secondary structure of the resulting protein, which in turn is recognized to determine the protein's interactions with other molecules. Therefore, in the SMVAD of the embodiments, such conservative substitutions can be made and are likely to have only a slight effect on their activity. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+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); tryptophan (-3.4). These values ​​can be used as a guide, and therefore, amino acid substitutions with a hydrophilicity value of 2 or less are preferred, those with a value of 1 or less are particularly preferred, and those with a value of 0.5 or less are even more particularly preferred. Therefore, any of the SMVADs described herein may be modified by substituting an amino acid with a different but homologous amino acid having a similar hydrophilicity value. Amino acids with a hydrophilicity of + / - 1.0 or + / - 0.5 points or less are considered homologous. Furthermore, it is assumed that SMVAD sequences may be modified by deletion, substitution, addition, or insertion of amino acids while maintaining their binding activity.

[0049] In certain embodiments, the human differentiation antigen group 6 binding molecule comprises one or more CDRs shown in SEQ ID NOs: 2-4, 6-8, 10-12, 14-16, 18-20, 22-24, 26-28, 30-32, 34-36, 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, and / or a variable region or CDR having one or more conserved or non-conserved amino acid changes in these SEQ ID NOs: 1-80, as well as the nucleic acid sequence encoding SEQ ID NOs: 1-80. Modification of the nucleic acid sequence encoding the amino acid sequence can result in a change to the amino acid sequence of the CDR or variable region. Nucleic acid sequences encoding a given CDR or variable region variant can be prepared by methods known in the art using the guidelines herein for specific sequences. These methods include, but are not limited to, site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and preparation by cassette mutagenesis of previously prepared nucleic acids encoding a CDR or variable region.

[0050] Simply put, to perform site-directed mutagenesis of DNA, the start DNA is first modified by hybridizing an oligonucleotide encoding the desired mutation onto a single strand of such start DNA. After hybridization, DNA polymerase is used to synthesize the entire second strand, using the hybridized oligonucleotide as a primer and the single strand of start DNA as a template. Thus, the oligonucleotide encoding the desired mutation is incorporated into the resulting double-stranded DNA.

[0051] PCR mutagenesis is also suitable for creating amino acid sequence variants of initiation CDRs (see, for example, Vallette et.al., (1989) Nucleic Acids Res. 17:723-733 (incorporated herein by reference)). Briefly speaking, when a small amount of template DNA is used as the initiation material for PCR, primers that are slightly different in sequence from the corresponding region in the template DNA can be used to generate a relatively large amount of specific DNA fragments that differ from the template sequence only at positions where the primer differs from the template.

[0052] Another method for preparing variants, cassette mutagenesis, is based on the technique described below: Wells et al., (1985) Gene 34:315-323 (incorporated herein by reference). The starting material is a plasmid (or other vector) containing the start CDR or variant region DNA to be mutated. The codon(s) in the start DNA to be mutated are identified. There must be specific restriction enzyme sites on both sides of the identified mutation site(s). If such restriction sites are not present, they can be generated using the oligonucleotide-mediated mutagenesis method described above to introduce them into the appropriate positions in the start polypeptide DNA. The plasmid DNA is cleaved at these sites to linearize it. A double-stranded oligonucleotide encoding the DNA sequence between the restriction sites but containing the desired mutation(s) is synthesized using a standard procedure, and the two strands of the oligonucleotide are synthesized separately and then hybridized together using a standard method. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 5' and 3' ends compatible with the ends of a linearized plasmid, allowing for direct ligation of the plasmid. This plasmid currently contains a variant DNA sequence.

[0053] Alternatively or additionally, a desired amino acid sequence encoding a CDR variant or variable region variant can be determined, and nucleic acid sequences encoding such amino acid sequence variants can be synthetically generated. Conservative modifications can be made to the amino acid sequences of the CDR or variable region. Naturally occurring residues are classified into classes based on common side-chain properties.

[0054] (1) Hydrophobic: norleucine, met, ala, val, leu, ile, (2) Neutral hydrophilic: cysteine, serine, threonine, (3) Acidic: asp, glu, (4) Basicity: asn, gln, his, lys, arg, (5) Residues that affect chain orientation: glycine, proline, and (6) Aromatics: trp, tyr, phe. Conservative substitution would inevitably involve replacing one member of a particular antibody, variable region, or CDR (e.g., SEQ ID NOs. 1-80) with another member of the same class.

[0055] For the expression of the human CD6-binding molecules disclosed herein, the expression vector(s) encoding SMVAD can be transfected into host cells using standard methods. The various forms of the term “transfection” are intended to encompass the wide variety of methods commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells (e.g., electroporation, calcium phosphate precipitation, DEAE dextran transfection, etc.).

[0056] In certain embodiments, the expression vector used to express the human CD6-binding molecule of the present invention is a viral vector (e.g., a retroviral vector). Such viral vectors can be used to generate a stably transduced cell line (e.g., a continuous source of differentiation antigen group 6-binding molecules). In some embodiments, GPEX gene product expression technology (Catalent, Somerset, New Jersey) is used to generate differentiation antigen group 6-binding molecules (and stable cell lines expressing differentiation antigen group 6-binding molecules). In certain embodiments, the expression technologies described in WO0202783 and WO0202738 (both of which are incorporated herein by reference in their entirety) are used.

[0057] Examples of mammalian host cells for expressing the human CD6-binding molecule of the present invention include PER. Differentiation Antigen Group 6® cells (Crucell, Netherlands), Chinese hamster ovary (CHO) cells (e.g., dhfr-CHO cells, used with a DHFR selection marker, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220 (e.g., RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621)), NSO myeloma cells, COS cells, and SP2 cells. When a recombinant expression vector encoding the human CD6-binding molecule is introduced into mammalian host cells, antibodies are generally produced by culturing the host cells for a sufficient time to express the human CD6-binding molecule within the host cells, or more preferably, for a sufficient time to secrete nanobodies into the culture medium in which the host cells grow. The human CD6-binding molecule can be recovered from the culture medium using standard protein purification methods.

[0058] In certain embodiments, the human differentiation antigen 6 binding molecules of the present invention (e.g., nanobodies or dual nanobodies) are useful in immunoassays for detecting or quantifying human differentiation antigen 6 in a sample (e.g., a purified blood sample from a subject). In some embodiments, immunoassays for differentiation antigen 6 typically involve incubating a biological sample in the presence of a detectably labeled antibody or antibody fragment of the present invention that is selectively capable of binding to differentiation antigen 6, and detecting the labeled peptide or antibody bound in the sample. Various clinical assay procedures are well known in the art.

[0059] This disclosure provides an immunoassay method for determining the presence, amount, or concentration of human differentiation antigens 6 in a test sample. Such a method may use any suitable assay known in the art. Examples of such assays include, but are not limited to, immunoassays (e.g., sandwich immunoassays (e.g., monoclonal-polyclonal sandwich immunoassays, e.g., radioisotope detection (radioimmunosystay (RIA)), and enzyme detection (enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assay, R&D Systems, Minneapolis, Minnesota)), competitive inhibition immunoassays (e.g., forward and reverse), fluorescence polarization immunoassay (FPIA), enzyme amplification immunoassay method (EMIT), ARCHITECT assay (ABBOTT), bioluminescence resonance energy transfer (BRET), and homogeneous chemiluminescence assays.

[0060] Human CD6-binding molecules can be captured on beads or nitrocellulose, or on any other solid support capable of immobilizing soluble proteins (e.g., magnetic beads). Next, a sample containing human CD6 is attached to the support, and then washed with a suitable buffer to remove unbound proteins. A second detectably labeled molecule (e.g., antibody or peptide) capable of binding to human CD6-binding molecules can be attached to the solid support, and then washed twice with buffer to remove unbound molecules. The amount of unbound label on the solid support can then be detected by a known method.

[0061] The detection of human CD6-binding molecules can be achieved by binding them to an enzyme used in enzyme-linked immunosorbent assay (EIA) or enzyme-linked immunosorbent assay (ELISA). The bound enzyme reacts with the exposed substrate to produce a chemical moiety that can be detected, for example, by spectrophotometer, fluorescence assay, or visual means. Enzymes that can be used to detectably label human CD6-binding molecules according to the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase.

[0062] In some embodiments of the present invention, the human CD6 detected in the above assay may be present in the biological sample. Any sample containing human CD6 can be used. Preferably, the sample is a biological fluid (e.g., blood, brain tissue, serum, lymph, urine, cerebrospinal fluid, amniotic fluid, synovial fluid, tissue extract, or homogenate). However, the present invention is not limited to assays using only these samples, and it is possible for those skilled in the art to determine suitable conditions that allow the use of other samples.

[0063] In situ detection can be achieved by taking a histological specimen from a patient and providing such specimen with a combination of the labeled human CD6-binding molecules of this disclosure. The human CD6-binding molecules are preferably provided by applying or superimposing the labeled CD6-binding molecules onto a biological sample (e.g., brain tissue). By using such a procedure, it is possible to determine not only the presence of CD6 but also the distribution of CD6 in the test tissue.

[0064] In certain embodiments, this specification provides a kit for detecting human differentiation antigen group 6, comprising a human differentiation antigen group 6 detection molecule. Such a kit may include any of the immunodiagnostic reagents described herein and may further include instructions for use regarding the use of the immunodiagnostic reagent in an immunoassay for determining the presence of human differentiation antigen group 6 in a test sample. The kit may also include other reagents (e.g., buffers, salts, enzymes, enzyme cofactors, substrates, detection reagents, etc.) necessary to perform the diagnostic assay or facilitate quality control evaluation. Other components (e.g., buffers and solutions for the separation and / or processing of the test sample, e.g., pretreatment reagents) may also be included in the kit. The kit may further include one or more other controls. One or more components of the kit may be lyophilized, in which case the kit may further include reagents suitable for reconstitution of the lyophilized components.

[0065] The various components of the kit may be supplied in suitable containers (e.g., microtiter plates) as needed. The kit may further include containers for holding or storing samples (e.g., sample containers or cartridges). Optionally, the kit may also include reaction vessels, mixing vessels, and other components to facilitate the preparation of reagents or test samples. The kit may also include one or more instruments to assist in obtaining test samples (e.g., syringes, pipettes, forceps, measuring spoons, etc.).

[0066] References: Aleandri,S.,et al.Dynamic Light Scattering of Biopharmaceutics-Can Analytical Performance Be Enhanced by Laser Power?Pharmaceutics(2018)10:94. Crauwels,M.,et al.Reshaping nanobodies for affinity purification on protein a.N Biotechnol.(2020)57:20-28. Garner,L.I.,et al.,CD6 monoclonal antibodies differ in epitope,kinetics and mechanism of action.Immunology(2018)155:273-282. Nguyen,V.K.,et al.Camel heavy-chain antibodies:diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire.EMBO J.(2000)19:921-30. Sydow,J.F.,et al.Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions.PLoS One(2014)9(6):e100736. Vincke,C.,et al.General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold.J Biol Chem.(2009)284:3273-3284[doi:10.1074 / jbc.M806889200] All publications and patents referenced herein are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention described herein will be obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the invention is described in relation to certain preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described form for carrying out the invention which will be obvious to those skilled in the art in chemistry, medicine, molecular biology or related fields are intended to fall within the scope of the following claims. [Brief explanation of the drawing]

[0067] [Figure 1] The amino acid sequence (SEQ ID NO: 1) of the clone 2G1 CD6 nanobody VHH sequence, including CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3), and CDR3 (SEQ ID NO: 4), is shown. [Figure 2] This shows an exemplary step-by-step procedure used to generate CD6 nanobodies. [Figure 3] This study demonstrates that purified CD6 clone 2G1 binds to human CD6 with similar affinity and is detectable at 0.1 pM. [Figure 4] This study demonstrates that cloned 2G1 nanobodies bind to T cell lines (CD6+) in a dose-dependent manner. [Figure 5] This paper presents an exemplary design of a novel divalent anti-CD6 nanobody based on next-generation CD6-ADC. [Figure 6A] This demonstrates that the designed CD6 nanobody selectively binds to CD6 on T cells. [Figure 6B] This demonstrates that the designed CD6 nanobody selectively binds to CD6 on T cells. [Figure 7] This study demonstrates that divalent CD6 nanobodies are internalized in T cells (HuT78). [Figure 8] This study demonstrates that next-generation CD6-ADCs kill T-cell lymphoma cells more effectively than first-generation CD6-ADCs. [Figure 9]This study demonstrates that MMAE (monomethyl auristatin) bound to the CD6 nanobody dimer (2G1-2G1) inhibits the proliferation of human T cells (HH cell line). [Figure 10] A shows the amino acid sequence (SEQ ID NO: 5) of the clone A1CD601 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 6), CDR2 (SEQ ID NO: 7), and CDR3 (SEQ ID NO: 8). B shows the amino acid sequence (SEQ ID NO: 9) of the clone CD601HH1 nanobody VHH sequence containing CDR1 (SEQ ID NO: 10), CDR2 (SEQ ID NO: 11), and CDR3 (SEQ ID NO: 12). C shows the amino acid sequence (SEQ ID NO: 13) of the clone CD601HH2 nanobody VHH sequence containing CDR1 (SEQ ID NO: 14), CDR2 (SEQ ID NO: 15), and CDR3 (SEQ ID NO: 16). D shows the amino acid sequence (SEQ ID NO: 17) of the clone CD601HH3 nanobody VHH sequence containing CDR1 (SEQ ID NO: 18), CDR2 (SEQ ID NO: 19), and CDR3 (SEQ ID NO: 20). [Figure 11] A shows the amino acid sequence (SEQ ID NO: 21) of the clone CD601HH4 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 23), and CDR3 (SEQ ID NO: 24). B shows the amino acid sequence (SEQ ID NO: 25) of the clone CD601HH5 nanobody VHH sequence containing CDR1 (SEQ ID NO: 26), CDR2 (SEQ ID NO: 27), and CDR3 (SEQ ID NO: 28). C shows the amino acid sequence (SEQ ID NO: 29) of the clone CD601HH6 nanobody VHH sequence containing CDR1 (SEQ ID NO: 30), CDR2 (SEQ ID NO: 31), and CDR3 (SEQ ID NO: 32). D shows the amino acid sequence (SEQ ID NO: 33) of the clone CD601HH7 nanobody VHH sequence containing CDR1 (SEQ ID NO: 34), CDR2 (SEQ ID NO: 35), and CDR3 (SEQ ID NO: 36). [Figure 12]A shows the amino acid sequence (SEQ ID NO: 37) of the clone CD601HH8 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 38), CDR2 (SEQ ID NO: 39), and CDR3 (SEQ ID NO: 40). B shows the amino acid sequence (SEQ ID NO: 41) of the clone CD601HH9 nanobody VHH sequence containing CDR1 (SEQ ID NO: 42), CDR2 (SEQ ID NO: 43), and CDR3 (SEQ ID NO: 44). C shows the amino acid sequence (SEQ ID NO: 45) of the clone CD601HH10 nanobody VHH sequence containing CDR1 (SEQ ID NO: 46), CDR2 (SEQ ID NO: 47), and CDR3 (SEQ ID NO: 48). D shows the amino acid sequence (SEQ ID NO: 49) of the clone CD601HH11 nanobody VHH sequence containing CDR1 (SEQ ID NO: 50), CDR2 (SEQ ID NO: 51), and CDR3 (SEQ ID NO: 52). [Figure 13] A shows the amino acid sequence (SEQ ID NO: 53) of the clone CD601HH12 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 54), CDR2 (SEQ ID NO: 55), and CDR3 (SEQ ID NO: 56). B shows the amino acid sequence (SEQ ID NO: 57) of the clone CD601HH13 nanobody VHH sequence containing CDR1 (SEQ ID NO: 58), CDR2 (SEQ ID NO: 59), and CDR3 (SEQ ID NO: 60). C shows the amino acid sequence (SEQ ID NO: 61) of the clone CD601HH14 nanobody VHH sequence containing CDR1 (SEQ ID NO: 62), CDR2 (SEQ ID NO: 63), and CDR3 (SEQ ID NO: 64). D shows the amino acid sequence (SEQ ID NO: 65) of the clone CD601HH15 nanobody VHH sequence containing CDR1 (SEQ ID NO: 66), CDR2 (SEQ ID NO: 67), and CDR3 (SEQ ID NO: 68). [Figure 14]A shows the amino acid sequence (SEQ ID NO: 69) of the clone CD601HH16 chimeric nanobody VHH sequence containing CDR1 (SEQ ID NO: 70), CDR2 (SEQ ID NO: 71), and CDR3 (SEQ ID NO: 72). B shows the amino acid sequence (SEQ ID NO: 73) of the clone CD601HH17 nanobody VHH sequence containing CDR1 (SEQ ID NO: 74), CDR2 (SEQ ID NO: 75), and CDR3 (SEQ ID NO: 76). C shows the amino acid sequence (SEQ ID NO: 77) of the clone CD601HH18 nanobody VHH sequence containing CDR1 (SEQ ID NO: 78), CDR2 (SEQ ID NO: 79), and CDR3 (SEQ ID NO: 80). [Figure 15] This report presents the results of a CD6-ADC assay based on the killing of T-cell lymphoma cells by humanized CD6 nanobodies. A next-generation CD6-ADC was developed by conjugating one of the humanized CD6 nanobodies (HH4) with MMAE. T-cell lymphoma cell lines HH were cultured for 72 hours with either a new CD6-ADC (HH14-MMAE) or a control (hIgG-MMAE) with a molecular weight of 0–128 nM. Cell killing was quantified using trypan blue to distinguish dead cells from live cells.

Claims

1. A composition comprising a human differentiation antigen group 6 (CD6) binding molecule, or one or more nucleic acid molecules encoding the human CD6 binding molecule, The aforementioned human CD6-binding molecule A) Sequence IDs 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78; or CDR1 amino acid sequences containing Sequence IDs 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78 having one or two conservative amino acid changes. B) CDR2 amino acid sequences including SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79 having one or two conservative amino acid changes, and C) The composition comprising a first single monomer variable antibody domain (SMVAD) comprising a CDR3 amino acid sequence comprising SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80; or a CDR3 amino acid sequence comprising SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80 having one or two conservative amino acid changes.

2. The composition according to claim 1, wherein the first SMVAD further comprises four framework regions, the four framework regions being camelid, humanized, or human framework regions.

3. The aforementioned human CD6-binding molecule further, D) SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78; or CDR1 amino acid sequences containing SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78 having one or two conservative amino acid changes. E) CDR2 amino acid sequences including SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79; or CDR2 amino acid sequences including SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79 having one or two conservative amino acid changes, and F) The composition according to claim 1, comprising a second SMVAD comprising SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80; or a CDR3 amino acid sequence comprising SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80 having one or two conservative amino acid changes.

4. The composition according to claim 3, wherein the human CD6 binding molecule further comprises a linker bound to both the first SMVAD and the second SMVAD.

5. The composition according to claim 3, wherein the one or more nucleic acid molecules include: i) a first nucleic acid sequence encoding the first SMVAD and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region; and ii) a second nucleic acid sequence encoding the second SMVAD and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

6. The composition according to claim 1, wherein the first SMVAD comprises the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, or 77, or the amino acid sequence shown in SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, or 77 having deletions and / or conservative amino acid changes of SEQ ID NOs: 1, 2, 3, or 4.

7. The composition according to claim 1, wherein the human CD6-binding molecule further comprises a CH2 double chain constant region and / or a CH3 double chain constant region.

8. The composition according to claim 1, wherein the CH2 and / or CH3 heavy chain constant region is from a camelid, a humanized, or human.

9. The composition according to claim 1, wherein the human CD6-binding molecule is conjugated with a cytotoxic agent, and optionally the cytotoxic agent comprises monomethyl auristatin (MMAE).

10. Furthermore, the composition according to claim 1, comprising a physiologically acceptable buffer.

11. The composition according to claim 1, wherein the composition comprises one or more nucleic acid molecules, and optionally further comprises an expression vector, wherein one or more nucleic acid sequences are present within the expression vector.

12. The composition according to claim 1, wherein the composition comprises the human CD6-binding molecule.

13. The CDR1 amino acid sequence includes SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78. The CDR2 amino acid sequence includes SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79. The composition according to claim 1, wherein the CDR3 amino acid sequence includes sequence numbers 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80.

14. A method for treating or preventing T cell-related diseases or conditions, The process involves treating a subject with a composition comprising a human differentiation antigen group 6 (CD6) binding molecule, or with an expression vector comprising one or more nucleic acid molecules encoding the CD6 binding molecule, as enumerated in any one of claims 1 to 13. The method wherein the subject is suffering from or suspected of developing a T cell-related disease or condition.

15. The method according to claim 14, wherein the T-cell-related disease includes cancer, and optionally, the cancer includes T-cell lymphoma.

16. The method according to claim 14, wherein the T-cell-related disease includes acute respiratory distress syndrome (ARDS), cytokine release syndrome targeting Covid-19, or acute graft-versus-host disease (aCGDH).

17. The method according to claim 14, wherein the T-cell-related disease includes lupus nephritis, uncontrolled asthma, psoriasis, or multiple sclerosis.

18. The method according to claim 14, wherein the first SMVAD further comprises four framework regions, the four framework regions being camelid, humanization, or human framework regions.

19. The aforementioned human CD6-binding molecule further, D) SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78; or CDR1 amino acid sequences containing SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78 having one or two conservative amino acid changes. E) CDR2 amino acid sequences including SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79; or CDR2 amino acid sequences including SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79 having one or two conservative amino acid changes, and The method according to claim 14, comprising a second SMVAD comprising F) SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80; or a second SMVAD comprising a CDR3 amino acid sequence having one or two conservative amino acid changes, comprising SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80.

20. The method according to claim 19, wherein the human CD6 binding molecule further includes a linker bound to both the first SMVAD and the second SMVAD.

21. The method according to claim 19, wherein the one or more nucleic acid molecules include: i) a first nucleic acid sequence encoding the first SMVAD and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region; and ii) a second nucleic acid sequence encoding the second SMVAD and optionally further encoding a CH2 heavy chain constant region and / or a CH3 heavy chain constant region.

22. The method according to claim 14, wherein the first SMVAD comprises the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence shown in SEQ ID NO: 1 having one, two, three, or four deletions and / or conservative amino acid changes.

23. The method according to claim 14, wherein the human CD6-binding molecule further comprises a CH2 double chain constant region and / or a CH3 double chain constant region.

24. The method according to claim 14, wherein the CH2 and / or CH3 heavy chain constant region is from a camelid, a humanized, or human.

25. The method according to claim 14, wherein the human CD6 binding molecule comprises at least an antigen-binding portion of a clone 2G1 CD6 nanobody.

26. The method according to claim 14, wherein the composition further comprises a physiologically acceptable buffer.

27. The method according to claim 14, wherein the composition comprises the expression vector, and one or more nucleic acid sequences are present within the expression vector.

28. The method according to claim 14, wherein the composition comprises the human CD6-binding molecule.

29. The CDR1 amino acid sequence includes SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, or 78. The CDR2 amino acid sequence includes SEQ ID NOs: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, or 79. The method according to claim 14, wherein the CDR3 amino acid sequence includes SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or 80.

30. A method for detecting human differentiation antigen group 6 (CD6) in a sample, a) Contacting a sample with a human CD6 binding molecule according to any one of claims 1 to 13, wherein the sample is suspected to contain human CD6, and if the human CD6 binding molecule is present in the sample, it forms a complex with the human CD6, and further, the method b) The method comprising detecting the presence or absence of the complex in the sample.

31. The method according to claim 30, wherein the sample is derived from a subject suffering from or suspected of developing a T cell-related disease or condition.

32. The method according to claim 30, wherein the human CD6-binding molecule includes a detectable label.

33. The method according to claim 30, further comprising contacting the sample with a conjugate molecule capable of binding to the human CD6 binding molecule, wherein the conjugate molecule includes a detectable label.

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