Humanized chimeric antigen receptors targeting B-cell receptors in chronic lymphocytic leukemia and uses thereof

Abstract

The present invention relates to R110 mutant immunoglobulin lambda variable 3-21 (IGLV3-21 R110 The present invention provides a humanized chimeric antigen receptor (CAR) that targets the B cell receptor (BCR) of CLL cells characterized by a nucleotide sequence encoding the CAR, a vector containing the same, a pharmaceutical composition, and instructions for use.

Description

[Technical Field]

[0001] The present invention provides humanized chimeric antigen receptors (CARs) for the treatment of chronic lymphocytic leukemia (CLL). These CARs contain the R110 mutant immunoglobulin lambda variable 3-21 (IGLV3-21 R110 ) targeting the B cell receptor (BCR) of CLL cells, which are characterized by

[0002] The present invention also provides kits comprising nucleic acid sequences encoding the above-described CARs, vectors containing same, cells expressing same, pharmaceutical compositions, and instructions for use.

[0003] Background of the Invention Adoptive immunotherapy using chimeric antigen receptor (CAR)-redirected immune effector cells has emerged as a promising approach for the treatment of leukemia and lymphoma resulting from malignant transformation of B-lineage cells. Since the approval of CAR-T cell therapy, including the use of CARs directed against the common B-cell antigen CD19, the response rate, long-term outcomes, and quality of life of patients with certain B-cell leukemias and lymphomas, such as acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and mantle cell lymphoma, have been significantly improved (e.g., Maude SL, Frey N, Shaw PA et al. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia; N Engl J Med 2014;371:1507-15017).

[0004] The most common type of leukemia in Western countries is chronic lymphocytic leukemia, which usually occurs in older patients, and men are at twice the risk of developing CLL compared to women (Kipps TJ, Stevenson FK, Wu CJ, Croce CM, Packham G, Wierda WG, et al. Chronic lymphocytic leukemia. Nat Rev Dis Primers (2017) 3:1-12). CLL is a heterogeneous B-lymphocyte-derived malignancy resulting from the clonal expansion of a CD5-positive subpopulation of B lymphocytes that progressively accumulates in the bone marrow, lymph nodes, peripheral blood, and spleen (Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med. 1995; 333: 1052-1057).

[0005] As reviewed by Burger and Chiorazzi (Burger JA, Chiorazzi N. B cell receptor signaling in chronic lymphocytic leukemia. Trends Immunol 2013; 34: 592-601), clinical and biological evidence indicates that the BCR is one of the key factors in the clonal selection and survival of CLL cells.

[0006] BCRs are multiprotein structures composed of noncovalently associated antigen-binding and signaling subunits. The antigen-binding subunits consist of membrane immunoglobulins containing two identical heavy chains and two identical light chains, each with one constant domain per light chain and three constant domains per heavy chain. Each heavy chain associates with a light chain to form an antigen-binding site. Each light and heavy chain contains a variable domain that also forms the antigen-binding site. Immunoglobulin genes encoded by the Igh, Igl, and Igk loci contain multiple V (variable), D (diversity), and J (joining) gene segments upstream of one or more constant exons. In developing B cells, immunoglobulin gene rearrangements randomly combine V, D, and J gene segments to create a complete V exon at the Igh locus and V and J gene segments at either the Igk or Igl locus. Through combinatorial combination of gene segments, binding diversity, and random heavy and light chain pairing, each individual B cell precursor generates a unique, nearly unique antigen-binding subunit, whose antigen-binding affinity can be further refined by somatic hypermutation (SHM).

[0007] The signaling portion of the BCR consists of a disulfide-linked heterodimer of Igα and Igβ (CD79a / CD79b) proteins, each of which contains a single immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail that initiates signal transduction after BCR aggregation upon antigen binding (Flaswinkel, H., Reth, M., 1994. Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor. EMBO J. 13, 83-89).

[0008] Antigen binding rapidly activates the Src family kinase Lyn, resulting in the phosphorylation of Igα / Igβ. This initiates the formation of a large signaling complex on the cytoplasmic side of the membrane, consisting of the BCR, various tyrosine kinases, adaptor proteins, and signaling enzymes. Proximal BCR signaling is mediated by the protein tyrosine kinase Syk (spleen tyrosine kinase), which recruits Syk to phosphorylated ITAMs of Igα and Igβ. Signal propagation occurs through the association of Syk with the adaptor protein SLP65 and its downstream signaling enzymes, Bruton's tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). Signals emanating from the signaling complex activate downstream pathways, including calcium mobilization, phosphoinositide 3-kinase (PI3K), nuclear factor kappa B (NF-κB), nuclear factor of activated T cells (NF-AT), mitogen-activated protein kinase (MAPK), and rat sarcoma (RAS) signaling pathways (Burger JA and Chiorazzi N, 2013, sa).

[0009] Chronic activation of mature B cells via the B cell receptor has been shown to be a critical process in the formation and development of CLL (Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G. B-cell receptor signaling in chronic lymphocytic leukemia Blood. 2011;118:4313-4320). This is consistent with studies using the EBV protein LMP2A as a constitutively active BCR surrogate, which showed that the development of the murine B1 subset is strongly dependent on prolonged BCR stimulation (Casola S, Otipoby KI, Alimzhanov M, et al. B cell receptor signal strength determines B cell fate. Nat Immunol 2004;5:317-27). Furthermore, antigen-independent, autonomous signaling in primary CLL B cells resulting from the interaction of two adjacent BCRs on the cell has been identified as a key driver of CLL pathogenesis, leading to increased tyrosine phosphorylation of BCR-proximal signaling molecules, resulting in cyclic signaling and Ca2+ upregulation. 2+ This leads to increased recruitment (Duehren-von Minden M et al. Chronic lymphocytic leukemia is driven by antigen-independent cell-autonomous signaling. Nature. 2012; 489: 309-313).

[0010] It is well documented that the protein kinase Syk is constitutively phosphorylated by sustained BCR signaling, and several studies have revealed that other key molecules in the signaling pathway downstream of BCR ligation in normal B cells, such as PKC, phosphoinositide 3-kinase, and mitogen-activated protein kinase p38, are also constitutively activated in B-CLL cells, resulting in deregulation of the activity or expression of several pro-survival molecules and downstream pathways (Gobessi S, Laurenti L, Longo PG, Carsetti L, Berno V, Sica S et al. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia 2009; 23: 686-697.; Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C et al. Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase C delta. Blood 2002; 100: 3741-3748., Plate JM. PI3-kinase regulates survival of chronic lymphocytic leukemia B-cells by preventing caspase 8 activation. Leuk Lymphoma 2004; 45: 1519-1529., Sainz-Perez A, Gary-Gouy H, Portier A, Davi F, Merle-Beral H, Galanaud P et al.High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia. Leukemia 2006; 20: 498-504.). .

[0011] Constitutively activated signaling pathways such as NF-kB or PI3K / AKT have been shown to cause the transcription and overexpression of key anti-apoptotic proteins, in particular B-cell lymphoma 2 (Bcl-2) and several members of the inhibitor of apoptosis protein (IAP) family (Loeder S et al. A novel paradigm to trigger apoptosis in chronic lymphocytic leukemia. Cancer Res. 2009; 69: 8977-8986). In addition to Bcl-2 itself, it is well established that Mcl-1 plays a crucial role in impairing apoptosis in CLL cells, and BCR signaling has been reported to upregulate Mcl-1 expression through the PI3K / AKT pathway (Petlickovski A, Laurenti L, Li X, Marietti S, Chiusolo P, Sica S, Leone G, Efremov DG. Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells. Blood. 2005; 105: 4820-4827).

[0012] Various aspects of the BCR have been recognized to identify major CLL disease subtypes. For example, the level of somatic hypermutation within the variable region of the BCR immunoglobulin heavy chain (IGHV) has been used as a prognostic marker for decades. CLL patients with mutated IGHV genes (M-CLL), i.e., CLL patients showing less than 98% IGHV gene identity with the closest germline, generally have a more indolent disease course than CLL patients with unmutated IGHV genes (U-CLL), which have germline identity of 98% or more. However, exceptions to this rule have been observed, in which mutant IGHV gene status cannot be correlated with a specific disease course. For example, cases with IGHV3-21 genes had one of the worst clinical outcomes, despite the majority expressing mutant BCRs. Different, but IGHV-determined, approaches have classified approximately 30% of CLL cases into distinct prognostically significant subsets, each with highly homogeneous biological features, clinical presentations, and outcomes. This classification is based on the observation that there are stereotypic BCRs that possess closely homologous heavy chain complementarity-determining region 3 (H-CDR3) sequences between mutated and unmutated cases.Following this approach, CLL cases characterized by mutated IGHV3-21 can be assigned to the so-called subset #2 (Stamatopoulos K, Belessi C, Moreno C, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenic implications and clinical correlations. Blood. 2007; 109(1): 259-270; Agathangelidis A., et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: A molecular classification with implications for targeted therapies. Blood. 2012; 119: 4467-4475).

[0013] Of note, IGHV3-21 usage by subset #2 is consistent with the immunoglobulin lambda variable 3-21 chain (IGLV3-21) with an acquired substitution of glycine by arginine at amino acid position 110 of the light chain. R110 ) is always observed in association with the expression of IGLV3-21 R110The arginine 110 (R110) of αβ is a single G>C substitution at the splice site between the immunoglobulin lambda J and constant genes. The presence of R110, along with the germline-encoded lysine 16 (K16) of one BCR and aspartates (D) 50 and 52 of the tyrosine-aspartic acid-serine-aspartic acid (YDSD) motif of an adjacent BCR, has been shown to enable BCR-BCR interactions and trigger cell-autonomous signaling (Figures 13 and 14; Minici, C. et al., Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukemia, Nature Comm. 2017;8:15746).

[0014] During the course of epigenetic, genomic, and transcriptomic characterization of a large cohort of CLL patients focusing on the BCR light chain, approximately 60% of patients were IGLV3-21 R110 It was revealed that the cases carried non-stereotypic BCRs, and subset #2 was identified as IGLV3-21 R110 It has been emphasized that this is only a minor subgroup of CLL characterized by the presence of light chain leukemia (Stamatopoulos B, Smith T, Crompot E, et al. The Light Chain lgLV3-21 Defines a New Poor Prognostic Subgroup in Chronic Lymphocytic Leukemia: Results of a Multicenter Study. Clin Cancer Res. 2018; 24(20): 5048-5057., Nadeu F, Royo R, Clot G, et al. IGLV3-21 R110identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics. Blood. 2021; 137(21): 2935-2946).

[0015] Of the four alleles of the IGLV3-21 gene identified in humans, the allele IGLV3-21 * 01 (IMGT / LIGM-DB accession number X71966) and IGLV3-21 * 04 (IMGT / LIGM-DB accession no. AC279208) encodes essential K16 and D50 and D52, the last two of which are identical to IGLV3-21 in most cases studied. R110 The motif contains a tyrosine at position 49 and a serine at position 51 (exemplary IGLV3-21 R110 (See Figure 14 for details.) However, IGLV3-21 R110 Functionally equivalent mutations in this motif, such as the replacement of tyrosine by phenylalanine or serine by threonine, have also been observed in CLL patients (Nadeu et al. 2021, sa). Interestingly, the allele IGLV3-21 * 01 and IGLV3-21 * 04 is present in very low numbers in B cells of healthy donors, but in patients studied by different groups, all IGLV3-21 genes are expressed as alleles IGLV3-21 * 01 or allele IGLV3-21 * 04, and these alleles are IGLV3-21 R110 It has been suggested that it may be mechanistically required for the development of related CLL.

[0016] IGLV3-21 R110 A CLL subgroup, also proposed to be named subset #2L, is associated with a very rapid disease course. R110The poor outcome of CLL cases is independent of IGHV mutation status or heavy chain properties. R110 is found in mutant CLL, such as subset #2 (see above), as well as in the context of different heavy chains, such as IGHV1-18, IGHV3-53, or IGHV3-64 (Nadeu et al. 2021, supra), and therefore IGLV3-21 R110 defines a group of CLLs that is not limited by traditional subset classifications based on empirically defined epigenetic stereotypes, nor by IGHV mutation status. Furthermore, the essential role of R110 as a CLL driver mutation has been confirmed by site-directed mutagenesis experiments, and IGLV3-21 R110 IGLV3-21 G110 It was revealed that restoring BCR to its normal state abolished its autonomous signaling ability (Stamatopoulos B, Smith T, Crompot E, et al. 2018, sa).

[0017] Time to first treatment (TTFT) and overall survival (OS) with IGLV3-21 R110 In a study correlating the presence of harboring BCR, IGLV3-21 R110 In patients expressing non-IGLV3-21 R110 The values ​​were significantly shorter than those in CLL patients, and IGLV3-21 R110 The urgent need for therapy for positive patients was emphasized (Nadeu F et al. 2021; sa).

[0018] However, CARs targeting common B cell antigens such as CD19 do not distinguish between healthy and malignant B cells, so patients experience persistent B cell depletion as long as anti-CD19 CAR T cells persist. Due to immunosuppression, inability to generate a vaccine response, high infection risk, and severe hematologic toxicity that causes off-target effects on the immune system, no CAR has yet proven suitable for the treatment of CLL (see, e.g., Porter DL, Levine BL, Kalos M et al. Chimeric Antigen Receptor-Modified T Cells in Chronic Lymphoid Leukemia; N Engl J Med 2011; 365: 725-733).

[0019] Therefore, IGLV3-21, which does not have the above-mentioned side effects, R110 A specific CAR for the treatment of positive CLL patients has not yet been found.

[0020] CARs are artificially engineered chimeric transmembrane proteins or polypeptides. The transmembrane domain anchors the CAR to the cell membrane of immune cells and connects the extracellular domain containing the antigen-binding domain with the cytoplasmic domain that provides the immune cell with effector function. Immune effector cells engineered to express the CAR gene redirect the cells to kill tumors expressing the surface antigen targeted by the CAR's antigen-binding domain. CARs have the ability to redirect immune cell specificity and reactivity to selected targets, independent of MHC, by utilizing the antigen-binding properties of antibodies. Antigen-binding domains are traditionally derived from monoclonal antibodies. WO 2019 / 008129 discloses a monoclonal antibody that can be used to remove CLL cells from blood samples as a candidate fusion protein with a T cell-specific activation domain to obtain a CAR. However, no therapeutic effect of such a CAR has been demonstrated. Furthermore, the antibody in WO 2019 / 008129 is expressed as an IgG antibody in a hybridoma cell line derived from a mouse host. A potential problem with using mouse monoclonal antibodies as the basis for CARs is that they may be recognized as foreign and elicit an immune response in patients. This immunogenicity can lead to poor patient persistence, and reactions such as the human anti-mouse antibody (HAMA) response may prohibit repeated administration of CAR-exposed cells.

[0021] From the above, IGLV3-21 R110 Treatment options that could improve the treatment of CLL in positive patients, with reduced risk of toxicity and immunogenic reactions, particularly CARs that are selective for healthy B cells and not cross-reactive, remain unknown but are needed.

[0022] Summary of the Invention In a first aspect, the present invention provides a humanized IGLV3-21 R110 A novel humanized CAR comprising a binding domain, a transmembrane domain, and a cytoplasmic domain, comprising humanized IGLV3-21 R110The above problem is solved by providing a humanized CAR, the binding domain of which comprises a light chain variable region (VL) including a light chain complementarity determining region 1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 1, a light chain complementarity determining region 2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 2, and a light chain complementarity determining region 3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region (VH) including a heavy chain complementarity determining region 1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 (H-CDR2) having the amino acid sequence of SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 6.

[0023] Such a humanized CAR is IGLV3-21 R110 This new CAR specifically binds to the BCR carrying the IGLV3-21 BCR, and unlike anti-CD19 CARs, mediates killing of malignant B cells without off-target killing of healthy B cells in CLL patients. R110 While recognizing the existence of the IGHV3-21 / IGLV3-21 R110 "Subset #2 CLL," characterized by a BCR comprising the combination of: R110 It is also possible to target any other disease including CLL.

[0024] In a second aspect, the present invention also relates to a polynucleotide encoding the humanized CAR of the present invention. Thus, in a third aspect, the present invention also relates to a vector comprising the nucleic acid sequence of the present invention.

[0025] In a fourth aspect, the present invention relates to a cell expressing a humanized CAR according to the present invention.

[0026] In a fifth aspect, the present invention relates to a pharmaceutical composition according to the invention.

[0027] In a sixth aspect, the present invention provides a method for the treatment of diseases, in particular IGLV3-21 R110The sixth aspect of the present invention relates to a method for treating a disease, in particular an IGLV3-21 positive patient, by administering a CAR, polynucleotide, vector, cell or pharmaceutical composition according to the present invention to a subject in need thereof. R110 The present invention relates to a method of treating CLL in a positive patient.

[0028] According to this sixth aspect, the CAR, polynucleotide, vector, cell or pharmaceutical composition described above can be administered together with an additional therapeutic agent. Specifically, the therapeutic agent can be Bruton's tyrosine kinase (BTK).

[0029] A further aspect provides a kit comprising a CAR, a polynucleotide, a vector, a cell or a pharmaceutical composition according to the invention.

[0030] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. However, the following references can provide those skilled in the art with general definitions of many of the terms used herein and can be referenced and used to the extent that such definitions are consistent with the meanings commonly understood in the art. Such references include, but are not limited to, Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), Hale & Marham, The Harper Collins Dictionary of Biology (1991), and Lackie et al., The Dictionary of Cell & Molecular Biology (3rd ed. 1999), and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, WB Saunders Company. Reference may be made to any additional technical resources available to those skilled in the art that provide definitions of the terms used herein that have meanings commonly understood in the art. For purposes of the present invention, the following terms are further defined:

[0031] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

[0032] "Autonomously active" BCRs are a special type of constitutively active BCR. While traditional activation is based on external antigens, autonomously active BCRs are activated by interactions with membrane structures on the surface of the same cell. Regarding the clinical manifestations of CLL, we have demonstrated interactions that induce autonomous activation between adjacent BCRs on the surface of the same cell (e.g., M. Duehren-von Minden et al.; Nature 2012).

[0033] IGLV3-21 R110 is the light chain variable region of the BCR that enables BCR-BCR interaction and induces autonomously active BCR. R110 are characterized by a sequence identity of more than 80% to the sequence represented by SEQ ID NO: 41, in both cases having an arginine instead of a glycine at position 110 of the above sequence.

[0034] The term "antibody," as used herein, is intended to refer to an immunoglobulin molecule, preferably an immunoglobulin molecule composed of four polypeptide chains: two heavy (H) chains and two light (L) chains, usually interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may contain, for example, three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs), that provide suitable anchorage for the CDRs.

[0035] As used herein, the term "complementarity determining region" (CDR; e.g., CDR1, CDR2, and CDR3) refers to the amino acid residues of the variable domain of a molecule capable of binding to an antigen, such as an antibody, antibody fragment, or single-chain variable fragment (scFv), the presence of which is necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Chothia and Lesk (J Mol Biol 196: 901-917 (1987)), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). Each complementarity-determining region comprises amino acid residues defined by IMGT. In some cases, a complementarity-determining region may also comprise amino acids from a CDR region defined according to Kabat and / or a hypervariable loop according to the Chothia numbering system.

[0036] The term "CAR" or "chimeric antigen receptor" as used herein is intended to refer to an artificial transmembrane protein that induces an antigen-induced response in immune cells. The immune cells modified by CARs can be, for example, naive T cells, central memory T cells, effector memory T cells, or natural killer (NK) cells. CARs typically comprise an extracellular domain and a cytoplasmic domain connected by a transmembrane domain. The extracellular domain comprises at least an antigen-binding domain.

[0037] Here, an "antigen-binding domain" is defined as a polypeptide portion of a CAR that recognizes an antigen. Specifically, the antigen-binding domain of the present invention is a polypeptide having an antigen-binding site comprising complementarity-determining regions (CDRs). The antigen-binding domain may comprise six CDRs and have an antigen-binding site corresponding to the heavy chain variable region (VH) and light chain variable region (VL) of a classical antibody. The VH of a classical antibody is generated by immunoglobulin gene rearrangement of V (variable), D (diversity), and J (joining) region gene segments, while the VL is generated by V and J gene segment rearrangement. Each VH and VL thus generated typically consists of three CDRs and up to four FRs, arranged from the amino terminus to the carboxy terminus, for example, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antigen-binding domain is usually a single-chain variable fragment (scFv) derived from an antibody, but can be based on other formats, including Fab, Fab', F(ab')2, VH and VL antibody chains (Fv fragments), full-length heavy and light chains, and linear antibodies.

[0038] The "antigen binding site" is typically found in one or more hypervariable regions, e.g., the CDR1, CDR2 and / or CDR3 regions. However, variable "framework" regions may also play an important role in antigen binding, e.g., by presenting the antigen binding site in the appropriate form for binding to the antigen.

[0039] As used herein, a "humanized" CAR comprises a humanized antigen-binding domain to reduce immunogenicity. A humanized form of a non-human (e.g., murine) antigen-binding domain is a genetically engineered chimeric antigen-binding domain, whose variable regions, VL and VH, contain minimal sequences derived from non-human immunoglobulins. The variable regions of the antigen-binding domain can be (i) CDR-grafted, in which the CDRs of the variable regions are non-human and one or more framework regions of the variable regions are human; (ii) amino acids in the framework regions of the non-human variable regions are partially replaced with human amino acid sequences by genetic engineering; or (iii) derived from a non-human source (e.g., a transgenic mouse with a heterologous immune system) in which the variable regions are based on human germline sequences. Thus, a humanized antigen-binding domain can be an antigen-binding domain having some or all of the CDRs derived from non-human immunoglobulins and one or more framework (FR) variable region framework sequences derived from humans. Similarly, a humanized VL can have at least one, two, or three CDRs from a non-human immunoglobulin and one, two, three, or four framework (FR) variable region framework sequences of human origin. Similarly, a humanized VH can have at least one, two, or three CDRs from a non-human immunoglobulin and one, two, three, or four framework (FR) variable region framework sequences of human origin. In some cases, human-derived variable region framework residues are replaced with corresponding non-human residues. In some cases, the humanized variable region can include individual residues that are not found in either the human-derived variable region or the non-human immunoglobulin CDR. When the antigen-binding domain comprises at least a portion of an immunoglobulin constant region (Fc), that portion is typically that of a human immunoglobulin.

[0040] "Percent (%) sequence identity" to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues, respectively, in a candidate sequence that are identical with the nucleic acid or amino acid residues, respectively, in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered part of the sequence identity. Alignment for the purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, using publicly available computer software such as, for example, BLAST (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990; 215:403-10), BLAST-2, ALIGN, LALIGN or Megalign (DNASTAR) software, or the Immunogenetic Information System® (IMGT®) DomainGapAlign tool (Ehrenmann F, Kaas Q, Lefranc MP. IMGT / 3D structure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucleic Acids Res. 2010; 38, D301-307). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

[0041] As used herein, a CAR that "specifically binds to" an antigen of interest, such as a tumor-associated polypeptide antigen target (herein IGLV3-21 R110) "specific for" or "specifically recognize" an antigen and can distinguish between such antigen and one or more reference antigens. In its most general form, "specific binding," "specifically binds to," "specific for" or "specifically recognizes" refers to the ability of a CAR to distinguish between an antigen of interest and unrelated antigens, for example, as determined according to one of the following methods: Such methods include, but are not limited to, flow cytometry, Western blot, ELISA test, RIA test, ECL test, IRMA test, immunohistological test, and peptide scan.

[0042] "Binding affinity" or "affinity" refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise specified, as used herein, "binding affinity" or "affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The dissociation rate constant, K D is usually the equilibrium association (k a ) and dissociation rate (k d The dissociation constant, K D " is commonly used to describe the affinity between a molecule (such as an antibody) and its binding partner (such as an antigen), i.e., how tightly the ligand binds to a particular protein. Ligand-protein affinity is influenced by non-covalent intermolecular interactions between the two molecules. The term "high affinity" refers to the affinity of a molecule / antibody to IGLV3-21 R110 10 positive CLL BCR -9 This means binding with an affinity (KD) of M or less (monovalent affinity). A molecule / antibody may have substantially greater affinity for a target antigen than other unrelated molecules. Affinity can be measured by common methods known in the art, for example, according to Example 1.

[0043] The "mutants" of CAR contemplated in the present invention are IGLV3-21 R110 It is a molecule that maintains the binding activity of the antigen-binding domain to the target antigen.

[0044] The term "cancer" refers to a physiological condition or disease in which cells divide uncontrollably, causing unregulated cell growth. A "tumor" contains one or more cancerous cells.

[0045] Detailed Description of the Invention First Aspect of the Invention—CAR The humanized CAR of the present invention is IGLV3-21 R110 CARs can specifically bind to BCRs bearing the CAR-binding domain and provide a therapeutic effect to the subject. CARs and their beneficial properties that enable therapeutic activity are described in more detail below.

[0046] According to a first aspect of the present invention, a humanized anti-IGLV3-21 R110 A humanized chimeric antigen receptor (CAR) comprising a binding domain, a transmembrane domain, and a cytoplasmic domain, the humanized IGLV3-21 R110 A CAR is provided, wherein the binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determining region 1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 1, a light chain complementarity determining region 2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 2, and a light chain complementarity determining region 3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 (H-CDR2) having the amino acid sequence of SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 6.

[0047] Preferred embodiments of the first aspect of the present invention are further characterized in more detail in Table 3 of the Examples.

[0048] Thus, a preferred embodiment of the first aspect of the present invention is a CAR characterized by any combination of a VL having an amino acid sequence selected from the list consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 and a VH having an amino acid sequence selected from the list of SEQ ID NO:11 and SEQ ID NO:12.

[0049] In the above-mentioned preferred embodiment of the first aspect of the present invention, CAR "H1" characterized by a VL having the amino acid sequence of SEQ ID NO: 7 and a VH having the amino acid sequence of SEQ ID NO: 11 is a first more preferred embodiment.

[0050] In the above preferred embodiment of the first aspect of the invention, CAR "H2" characterized by a VL having the amino acid sequence of SEQ ID NO: 7 and a VH having the amino acid sequence of SEQ ID NO: 12 is a second more preferred embodiment.

[0051] In the above preferred embodiment of the first aspect of the present invention, CAR "H3" characterized by a VL having the amino acid sequence of SEQ ID NO: 8 and a VH having the amino acid sequence of SEQ ID NO: 11 is a third more preferred embodiment.

[0052] In the above-mentioned preferred embodiment of the first aspect of the present invention, CAR "H4" characterized by a VL having the amino acid sequence of SEQ ID NO: 8 and a VH having the amino acid sequence of SEQ ID NO: 12 is a fourth more preferred embodiment.

[0053] In the above-mentioned preferred embodiment of the first aspect of the present invention, CAR "H5" characterized by a VL having the amino acid sequence of SEQ ID NO: 9 and a VH having the amino acid sequence of SEQ ID NO: 11 is a fifth more preferred embodiment.

[0054] In the above-mentioned preferred embodiment of the first aspect of the present invention, CAR "H6" characterized by a VL having the amino acid sequence of SEQ ID NO: 9 and a VH having the amino acid sequence of SEQ ID NO: 12 is a sixth more preferred embodiment.

[0055] In the above-mentioned preferred embodiment of the first aspect of the present invention, CAR "H7" characterized by a VL having the amino acid sequence of SEQ ID NO: 10 and a VH having the amino acid sequence of SEQ ID NO: 11 is a seventh more preferred embodiment.

[0056] In the above-mentioned preferred embodiment of the first aspect of the present invention, CAR "H8" characterized by a VL having the amino acid sequence of SEQ ID NO: 10 and a VH having the amino acid sequence of SEQ ID NO: 12 is an eighth more preferred embodiment.

[0057] The most preferred CARs of the aforementioned more preferred embodiments of the first aspect of the invention are those of the first and second more preferred embodiments (“H1” and “H2”).

[0058] The CAR of this first aspect of the invention is not limited to the specific peptide sequence provided. Rather, the invention encompasses variants. Those skilled in the art will appreciate that, with reference to this disclosure and previously available techniques and references, the CAR of IGLV3-21 R110 Functional variants of the CARs disclosed herein can be prepared, tested, and utilized, with the understanding that these variants that have the ability to bind to the -BCR and thereby kill B cells are within the scope of the present invention.

[0059] Variants can include, for example, CARs with at least one complementarity determining region (CDR) (hypervariable) and / or framework (FR) (variable) region / position altered relative to the peptide sequences disclosed herein.

[0060] As an example, one skilled in the art can use the sequences of CARs provided herein (e.g., in Table 3) to design variants that are within the scope of the present invention.

[0061] Furthermore, variants can be obtained by using one CAR of this first aspect of the invention as a starting point for optimization by diversifying one or more amino acid residues in the CAR, preferably amino acid residues in one or more CDRs, and screening the resulting collection of CAR variants.

[0062] The polypeptide variants of CAR binding domains described herein can be made while preserving the overall molecular structure of CAR.Considering the characteristics of individual amino acids, those skilled in the art will recognize some reasonable substitutions.The "conservative substitution" of amino acid substitution can be made based on, for example, polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic similarity of related residues.

[0063] For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions can typically be made within groups (a) through (d). Additionally, glycine and proline may be substituted for each other based on their ability to disrupt a-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine, are more commonly found in a-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan, and threonine are more commonly found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns. Some preferred substitutions can be made between the following groups: (i) S and T, (ii) P and G, and (iii) A, V, L, and I. Given the known genetic code and recombinant and synthetic DNA techniques, one of skill in the art can readily construct DNAs encoding conservative amino acid variants.

[0064] The CAR according to the first aspect of the invention may also comprise a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity to the amino acid sequence set forth by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.

[0065] The CAR according to the first aspect of the invention may also comprise a VH having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity to the amino acid sequence set forth by SEQ ID NO:11 or SEQ ID NO:12.

[0066] Humanized anti-IGLV3-21 CAR according to the first aspect of the invention R110 The binding domain is preferably an scFv. The order of VL and VH within the scFv can be VL-VH or VH-VL from the N-terminus to the C-terminus, but the order of VL-VH is preferred. The VL of the scFv is preferably linked to the VH via a suitable amino acid linker. Preferred scFvs of the CAR according to the first aspect of the present invention have the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

[0067] The CAR according to the first aspect of the present invention may further comprise a transmembrane domain derived from a type 1 transmembrane protein such as CD28, CD3ζ, CD4, CD8α or OX40. Preferably, the transmembrane domain is of human origin. Specifically, the transmembrane domain may be derived from human CD28.

[0068] The CAR according to the first aspect of the present invention may further comprise a spacer region. A spacer is often required to separate the antigen-binding domain from the membrane and provide access to the antigen and some flexibility. The spacer may be derived from IgG1 or IgG4, or extracellular CD28, CD4, or CD8. Specifically, the spacer may be the CH1-CH2-CH3 constant region of IgG1 or IgG4. More specifically, a more compact spacer such as an IgG1 or IgG4 hinge may be sufficient. It may be preferable that the spacer is of human origin.

[0069] According to the first aspect of the present invention, the cytoplasmic domain can comprise, consist essentially of, or consist of a signaling domain. The signaling domain may comprise one or more tyrosine-based activation motifs, preferably immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs act as specific adapters for downstream signaling proteins after phosphorylation of conserved tyrosine residues within the motif. The signaling domain may be derived from, for example, but not limited to, CD3ζ, FcεR1, DAP10, or DAP12. The FcεR1 signaling domain contains one ITAM, and the CD3ζ signaling domain contains three ITAMs, and they associate with the T cell receptor to generate a signal. The signaling domain may be or include a T cell signaling domain. The signaling domain may preferably be of human origin. Specifically, the signaling domain may be derived from CD3ζ.

[0070] The cytoplasmic domain may contain one or more additional costimulatory domains. Suitable costimulatory domains may include domains derived from CD2, CD27, CD28, 4-1BB (CD137), CD244, ICOS, or OX40, which may be useful alone or in any combination thereof. Thus, the cytoplasmic domain may contain a signal transduction domain alone, or a signal transduction domain in combination with one or more costimulatory domains. Specifically, the cytoplasmic domain may contain a CD3ζ signal transduction domain in combination with the costimulatory domains CD28 and 4-1BB.

[0071] The humanized CAR according to the first aspect of the present invention is IGLV3-21 R110 (SEQ ID NO: 41 and FIG. 14). Unlike the anti-CD19 CAR, the humanized anti-IGLV3-21 R110 CAR inhibits wild-type IGLV3-21 G110 Mutant and malignant IGLV3-21 R110 IGLV3-21 effectively distinguishes between mutants and R110positive and IGLV3-21 R110 This can be demonstrated using negative cell lines as well as primary CLL cells (Example 3, Figures 3, 4, 6, 9).

[0072] Additionally, as described herein, humanized anti-IGLV3-21 R110 CAR-based CAR T cells are IGLV3-21 R110 It has been shown to selectively kill CD19-positive cell lines and primary CLL cells, but not polyclonal non-malignant B cells, which are similarly susceptible to killing by anti-CD19 CAR T cells. R110 CAR, IGLV3-21 R110 A beneficial embodiment of the invention is that it specifically and selectively binds to positive B cells, thereby allowing selective killing of malignant cells while sparing healthy human B cells (Example 3; Figure 7).

[0073] Anti-IGLV3-21 derived from healthy donor T cells R110 Superior killing was observed with CAR-based CAR T cells (e.g., Example 4 and Figure 11), and even CAR T cells derived from CLL patients demonstrated efficient killing capabilities despite the known dysfunction of T cells in CLL (Example 4 and Figure 11).

[0074] Furthermore, CLL B cell killing was accompanied by elevated levels of immune effector cytokines (Examples 5 and 6, Figures 5, 8, 12).

[0075] In summary, the humanized anti-IGLV3-21 antibody according to the present invention R110 CAR T cells express IGLV3-21 R110 It has been demonstrated that the humanized CAR is highly suitable for precise targeting of positive B cells. This precision and low immunogenicity make the humanized CAR of the present invention a potential tool in the field of therapeutic treatment of CLL patients, who are characterized by a defective immune system due to the underlying disease.

[0076] IGLV3-21 above R110Beyond the definitions presented above, R110 is, in a currently preferred embodiment of this first aspect of the invention, further characterized by greater than 80% sequence identity to the sequence represented by SEQ ID NO:41, wherein said sequence has a lysine at position 16 and aspartic acids at positions 50 and 52. In a currently more preferred embodiment of this first aspect of the invention, there is a tyrosine or phenylalanine at position 49 and a serine or threonine at position 51. In an even more currently preferred embodiment of this first aspect of the invention, there is a tyrosine at position 49 and a serine at position 51.

[0077] Second Aspect of the Invention—Polynucleotides The present invention also relates to a polynucleotide encoding a CAR of the first aspect of the invention. A polynucleotide according to the present invention may comprise DNA or RNA.

[0078] The DNA molecules of the present invention are not limited to the sequences disclosed herein and also include variants thereof. DNA variants of the present invention can be described with reference to their physical properties in hybridization. Those skilled in the art will recognize that nucleic acid hybridization techniques can be used to identify DNAs with their complements, and, because DNA is double-stranded, their equivalents or homologs. It will also be recognized that hybridization can occur with less than 100% complementarity. However, given the appropriate selection of conditions, hybridization techniques can be used to distinguish DNA sequences based on their structural relatedness to specific probes. For guidance on such conditions, see Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & Slruhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

[0079] The structural similarity between two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions under which the two sequences hybridize with each other.As used herein, the term "stringency" refers to the degree to which the conditions disfavor hybridization.Stringent conditions highly disfavor hybridization, and only the most structurally related molecules hybridize with each other under such conditions.Conversely, non-stringent conditions favor the hybridization of molecules that exhibit a lower degree of structural relatedness.Therefore, the stringency of hybridization is directly correlated with the structural relationship of two nucleic acid sequences.The following relationship is useful for correlating hybridization with relatedness (where Tm is the melting temperature of a nucleic acid duplex): aT m =69.3+0.41(G+C)% B T of double-stranded DNA m decreases by 1°C for every 1% increase in the number of mismatched base pairs. c.(T m )μ2-(T m )μ1=18.5log 10 μ2 / μ1 where μ1 and μ2 are the ionic strengths of the two solutions.

[0080] The stringency of hybridization is a function of many factors, including the overall nucleotide concentration, ionic strength, temperature, probe size and the presence of the substance that breaks hydrogen bond.The factors that promote hybridization include high nucleotide concentration, high ionic strength, low temperature, longer probe size and the absence of the substance that breaks hydrogen bond.Hybridization is usually carried out in two stages: " binding " stage and " washing " stage.

[0081] Yet another class of polynucleotide variants within the scope of the present invention can be described with reference to the products they encode. Those skilled in the art will understand that nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein can be made using routine techniques to reflect the codon usage of any particular host organism in which the product will be expressed. These functionally equivalent polynucleotides are characterized by encoding the same peptide sequences found in SEQ ID NOS: 1-20 due to the degeneracy of the genetic code.

[0082] It will be appreciated that variants of the DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as fully synthetic DNA. Methods for efficiently synthesizing oligonucleotides ranging from 20 to approximately 150 nucleotides are widely available. See Ausubel et al., section 2.11, Supplement 21 (1993). Overlapping oligonucleotides can be synthesized and assembled in the manner first reported by Khorana et al., J. Mol. Biol. 72:209-217 (1971). See also Ausubel et al., supra, Section 8.2. Synthetic DNA is preferably designed with convenient restriction sites engineered at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.

[0083] As indicated, a method for generating mutants is to start with one of the DNAs disclosed herein, followed by site-directed mutagenesis. See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typical method, the target DNA is cloned into a single-stranded DNA bacteriophage vehicle. The single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide change. The complementary strand is synthesized, and the double-stranded phage is introduced into a host. Some of the resulting progeny will contain the desired mutation, which can be confirmed using DNA sequencing. Additionally, various methods are available to increase the probability that the progeny phage will be the desired mutant. These methods are well known to those of skill in the art, and kits for generating such mutants are commercially available.

[0084] As with the first aspect of the invention, this second aspect of the invention also has corresponding preferred embodiments.

[0085] In a first preferred embodiment of this second aspect of the invention, the invention relates to a DNA molecule encoding a CAR comprising the sequence represented by SEQ ID NO: 21 (for L-CDR1), SEQ ID NO: 22 (for L-CDR2), SEQ ID NO: 23 (for L-CDR3), SEQ ID NO: 24 (for H-CDR1), SEQ ID NO: 25 (for H-CDR2) and SEQ ID NO: 26 (for H-CDR3).

[0086] In a second preferred embodiment of this second aspect of the invention, the invention relates to a DNA molecule encoding a CAR comprising a VL sequence represented by any sequence selected from the list consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.

[0087] In a third preferred embodiment of this second aspect of the invention, the invention relates to a DNA molecule encoding a CAR comprising a VH sequence represented by any sequence selected from the list consisting of SEQ ID NO: 31 and SEQ ID NO: 32.

[0088] In a fourth preferred embodiment of this second aspect of the invention, the invention relates to a DNA molecule encoding a CAR comprising an scFv sequence represented by any sequence selected from the list consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

[0089] Third Aspect - Vector A fifth aspect of the invention relates to a vector comprising a polynucleotide according to the second aspect of the invention.

[0090] Such a vector can be used to introduce a polynucleotide into a host cell so as to express a CAR according to the first aspect of the invention.

[0091] The vector may be a plasmid or a viral vector, preferably a lentiviral or retroviral vector.

[0092] Methods for packaging polynucleotides into plasmids or viral vectors are known in the art.

[0093] Vectors can be used to transduce or transfect cells.

[0094] In addition to the gene of interest, the vector may encode a reporter gene that allows for identification, detection and / or selection of the expressed gene of interest in a given host cell using molecular biological and immunological methods known in the art.

[0095] Such a reporter gene may be a truncated epidermal growth factor receptor (EGFRt) for antibody staining and sorting of CAR-expressing cells in flow cytometry. For co-expression of truncated EGFR, the vector may further encode a viral self-cleaving peptide, such as T2A.

[0096] Fourth aspect - cells A fourth aspect of the present invention relates to a cell expressing a CAR according to the first aspect of the present invention. The cell can be an immune effector cell, such as a T cell, for example, a naive T cell, a central memory T cell, an effector memory T cell, a natural killer (NK) cell, or a cytokine-induced killer cell. Preferably, the cell can be a human T cell.

[0097] This fourth aspect also relates to an in vitro method of producing a cell expressing a CAR, comprising introducing under suitable conditions into an immune cell according to the fourth aspect of the invention a polynucleotide according to the second aspect of the invention or a vector according to the third aspect of the invention.

[0098] Fifth Aspect—Pharmaceutical Composition The fifth aspect of the present invention relates to a pharmaceutical composition comprising a CAR according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, a vector according to the third aspect of the present invention, or a cell according to the fourth aspect of the present invention, and a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers can be, for example, vehicles, excipients, diluents, and adjuvants. Acceptable carriers are non-toxic to recipients and include, but are not limited to, stabilizers, buffers such as phosphate, citrate, or other organic acids, saline, buffered saline, low-molecular-weight polypeptides, proteins such as serum albumin, any sterile biocompatible pharmaceutical carrier, dextrose, and water. Optionally, the pharmaceutical composition may contain additional pharmaceutically active polypeptides and / or compounds.

[0099] The pharmaceutical composition according to the fifth aspect of the present invention may be manufactured in a manner known in the art, for example by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0100] Once a pharmaceutical composition comprising a CAR of the first aspect of the invention, a polynucleotide of the second aspect of the invention, a vector of the third aspect of the invention or a cell of the fourth aspect of the invention formulated in an acceptable carrier has been prepared, it can be placed in a suitable container and labelled for treatment of a designated condition.

[0101] Such administration is usually achieved parenterally. Methods of parenteral delivery include topical, intra-arterial (directly into the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular, intravenous, intraperitoneal, or intranasal administration.

[0102] The preferred routes of administration are intravenous and intra-arterial (directly into the tumor).

[0103] Pharmaceutical compositions for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0104] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation, and such penetrants are generally known in the art.

[0105] Sixth Aspect - Medical Use A sixth aspect of the present invention relates to a CAR according to the first aspect of the present invention, a polynucleotide according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a cell according to the fourth aspect of the present invention or a pharmaceutical composition according to the fifth aspect of the present invention, for use in treating a disease. In particular, the sixth aspect of the present invention relates to a cell according to the fourth aspect of the present invention, for use in treating a disease.

[0106] Similarly, this sixth aspect of the present invention relates to a method of treating a disease, comprising administering to a subject in need thereof a CAR of the first aspect of the present invention, a polynucleotide of the second aspect of the present invention, a vector of the third aspect of the present invention, a cell of the fourth aspect of the present invention or a pharmaceutical composition of the fifth aspect of the present invention. In particular, the method according to the sixth aspect of the present invention may comprise administering to the subject a cell of the fourth aspect of the present invention.

[0107] A method according to a sixth aspect of the present invention comprises: i) providing a population of immune cells; ii) introducing into immune cells a polynucleotide according to the second aspect of the invention or a vector according to the third aspect of the invention; iii) culturing the immune cells under conditions that allow expression of the CAR; (iii) administering the cells to a subject; Preferably, the cells may be autologous or allogeneic.

[0108] According to this sixth aspect of the present invention, the disease can be chronic lymphocytic leukemia (CLL). Specifically, the disease to be treated is clinically defined CLL caused by abnormal proliferation of B cells with autonomously active BCR. More specifically, the disease is IGLV3-21 R110 It is a clinically definite form of CLL characterized by the abnormal proliferation of B cells with autonomously active BCRs that possess the IL-1 receptor.

[0109] As discussed in relation to the first aspect of the present invention, humanized anti-IGLV3-21 R110 CAR is IGLV3-21 by CAR T cells R110 It has been shown to specifically and selectively mediate cytotoxic killing of malignant B cells expressing a positive BCR, and therefore, the humanized anti-IGLV3-21 according to the present invention for precision therapy of CLL with CAR T cells. R110 The suitability of CAR has been demonstrated.

[0110] According to a sixth aspect of the present invention, a CAR according to the first aspect of the present invention, a polypeptide according to the second aspect of the present invention, a vector according to the third aspect of the present invention, a cell according to the fourth aspect of the present invention, or a pharmaceutical composition according to the fifth aspect of the present invention may be administered to a subject in combination with an additional therapeutic agent. Thus, a CAR, polypeptide, vector, cell, or pharmaceutical composition may be administered as a sole pharmaceutical agent or in combination with one or more additional therapeutic agents if the combination does not cause unacceptable side effects. This combination therapy includes not only the administration of a single pharmaceutical formulation containing a CAR, polypeptide, vector, cell, or pharmaceutical composition and one or more additional therapeutic agents, but also the administration of a CAR, polypeptide, vector, cell, or pharmaceutical composition of the present invention and each additional therapeutic agent in its own separate pharmaceutical formulation.

[0111] When separate formulations are used, treatment according to the sixth aspect of the invention with one or more additional therapeutic agents may be essentially simultaneous (e.g., concurrent) or may be staggered (e.g., sequential). In particular, treatment according to the present invention may be carried out in fixed or separate combinations with other anti-tumor agents, such as alkylating agents, antimetabolites, plant-derived anti-tumor agents, hormone therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and / or biological response modifiers, anti-angiogenic compounds, and other anti-tumor agents.

[0112] A preferred additional therapeutic agent for co-administration according to the sixth aspect of the invention is a Bruton's tyrosine kinase (BTK) inhibitor. Preferably, the BTK inhibitor may be administered in combination with cells according to the fourth aspect of the invention, which may be autologous.

[0113] Certain Bruton tyrosine kinase (BTK) inhibitors are commercially available. Ibrutinib is an inhibitor known to induce apoptosis in B-cell lymphoma and CLL cells (Hermann SE, Gordon AL, Hertlein E, et al. Bruton tyrosine kinase represents a promising therapeutic target for the treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. 2011; 117: 6287-6296). Additional BTK inhibitors include, for example, ACP-196 from Acerta Pharma BV and BGB-3111 from BeiGene, Co., Ltd. Preferably, the additional therapeutic agent is ibrutinib. As shown in the Examples herein, the impaired functionality of CAR T cells derived from CLL patients can be completely restored by subtherapeutic administration of ibrutinib (Example 4, Figure 11).

[0114] Further Aspects of the Invention - Kits The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition according to the fifth aspect of the invention, which containers may be accompanied by a notice, in a form prescribed by the governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

[0115] Preferred embodiments of the present invention are as follows: A. Humanized IGLV3-21 R110 A humanized chimeric antigen receptor (CAR) comprising a binding domain, a transmembrane domain, and a cytoplasmic domain, the humanized IGLV3-21 R110A CAR, wherein the binding domain comprises a light chain variable region (VL) comprising a light chain complementarity determining region 1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 1, a light chain complementarity determining region 2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 2, and a light chain complementarity determining region 3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determining region 2 (H-CDR2) having the amino acid sequence of SEQ ID NO: 5, and a heavy chain complementarity determining region 3 (H-CDR3) having the amino acid sequence of SEQ ID NO: 6.

[0116] B. A CAR according to embodiment A, characterized by any combination of a VL having an amino acid sequence selected from the list consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and a VH having an amino acid sequence selected from the list of SEQ ID NO:11 and SEQ ID NO:12.

[0117] C. A CAR according to embodiment B, characterized by a VL having the amino acid sequence of SEQ ID NO: 7 and a VH having the amino acid sequence of SEQ ID NO: 11, or a VL having the amino acid sequence of SEQ ID NO: 7 and a VH having the amino acid sequence of SEQ ID NO: 12.

[0118] D. A CAR according to any of the previous embodiments, characterized by a VL having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity to the amino acid sequence of the VL according to embodiment C, and at least 70%, 75%, 80%, 85%, 90%, 95%, 98% amino acid sequence identity to the amino acid sequence of the VH according to embodiment C.

[0119] E. Humanized anti-IGLV3-21 R110 CAR according to any of the previous embodiments, wherein the binding domain is an scFv.

[0120] The CAR according to embodiment E, wherein the F.scFv has the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0121] G. A polynucleotide encoding a CAR according to any of the previous embodiments.

[0122] H. A vector comprising a polynucleotide according to embodiment G.

[0123] I. A cell expressing a CAR according to any of embodiments A-F.

[0124] The cell according to embodiment I, which is a JT cell.

[0125] K. A pharmaceutical composition comprising a CAR according to any of embodiments A-F, a polynucleotide according to embodiment G, a vector according to embodiment H, or a cell according to embodiment I or J, and a pharmaceutically acceptable carrier.

[0126] L. A CAR according to any of embodiments A-F, a polynucleotide according to embodiment G, a vector according to embodiment H, a cell according to embodiment I or J, or a pharmaceutical composition according to embodiment K for use in treating a disease.

[0127] M. A method of treating a disease, comprising administering a CAR according to any of embodiments A-F, a polynucleotide according to embodiment G, a vector according to embodiment H, a cell according to embodiment I or J, or a pharmaceutical composition according to embodiment K to a subject in need thereof.

[0128] N. iv) providing a population of immune cells; v) introducing into immune cells a polynucleotide according to embodiment G or a vector according to embodiment H; vi) culturing the immune cells under conditions that allow expression of the CAR; vii) administering the cells of (iii) to a subject; The method according to embodiment M, comprising:

[0129] O. A CAR, polypeptide, vector, cell or pharmaceutical composition for use according to embodiment L, or a method according to embodiment M or N, wherein the disease is chronic lymphocytic leukemia (CLL).

[0130] P. A CAR, polypeptide, vector, cell or pharmaceutical composition for use according to embodiment L or O, or a method according to any of embodiments MO, wherein the CAR, polypeptide, vector, cell or pharmaceutical composition is administered in combination with an additional therapeutic agent.

[0131] Q. The CAR, polypeptide, vector, cell, or pharmaceutical composition according to embodiment P, wherein the additional therapeutic agent is a Bruton's tyrosine kinase (BTK) inhibitor.

[0132] R. A kit comprising a pharmaceutical composition according to embodiment K. [Brief explanation of the drawings]

[0133] [Figure 1] FIG. 1 is a schematic diagram of humanized anti-IGLV3-21R110CAR. [Figure 2A] FIG. 1 shows the lentiviral vector pJ2459_h1_R110-1BB for expression of humanized anti-IGLV3-21R110CAR. [Figure 2B] FIG. 1 shows the lentiviral vector pJ2487_h1_R110-1BB for expression of humanized anti-IGLV3-21R110CAR. [Figure 3A]

[0023] Figure 3D are representative photographs of cytotoxicity assays after 24 hours of co-incubation according to Example 3. Figure 3A shows an assay of anti-IGLV3-21R110 CAR T cells from a healthy donor expressing humanized scFv H1 (3A:CARHDh1) or scFv H2 (3B:CARHDh2), and OCI-LY1_wt cells (OCI wt) derived from a patient whose CLL is not characterized by an IGHV3-21R110-BCR (i.e., non-IGLV3-21R110CLL). As can be seen from a comparison of Figures 3A and 3D, anti-IGLV3-21R110 CAR T cells from a healthy donor with humanized scFv H1 and scFv H2 show selective killing of the IGLV3-21R110-expressing cell line OCI-LY1_R110, indicated by large bright clusters. [Figure 3B]

[0023] Figure 3E are representative photographs of cytotoxicity assays after 24 hours of co-incubation according to Example 3. Figure 3B shows an assay of anti-IGLV3-21R110 CAR T cells from a healthy donor expressing humanized scFv H1 (3A:CARHDh1) or scFv H2 (3B:CARHDh2), and OCI-LY1_wt cells (OCI wt) derived from a patient whose CLL is not characterized by an IGHV3-21R110-BCR (i.e., non-IGLV3-21R110CLL). As can be seen from a comparison of Figures 3B and 3E, anti-IGLV3-21R110 CAR T cells from a healthy donor with humanized scFv H1 and scFv H2 show selective killing of the IGLV3-21R110-expressing cell line OCI-LY1_R110, indicated by large bright clusters. [Figure 3C] Representative photographs of cytotoxicity assays after 24 hours of co-incubation according to Example 3. Figure 3C shows a control using non-transduced CAR T cells from a healthy donor. [Figure 3D]

[0023] Figure 3D is a representative photograph of a cytotoxicity assay after 24 hours of co-incubation according to Example 3. Figure 3A depicts the same assay using the generated IGLV3-21R110-expressing cell line OCI-LY1_R110 (OCIR110). As can be seen from a comparison of Figures 3A and 3D, healthy donor-derived anti-IGLV3-21R110 CAR T cells with humanized scFv H1 and scFv H2 show selective killing of the IGLV3-21R110-expressing cell line OCI-LY1_R110, as indicated by the large bright clusters. [Figure 3E] Figure 3B is a representative photograph of a cytotoxicity assay after 24 hours of co-incubation according to Example 3. Figure 3E depicts the same assay using the generated IGLV3-21R110-expressing cell line OCI-LY1_R110 (OCIR110). As can be seen from a comparison of Figures 3B and 3E, anti-IGLV3-21R110 CAR T cells from a healthy donor with humanized scFv H1 and scFv H2 show selective killing of the IGLV3-21R110-expressing cell line OCI-LY1_R110, as indicated by the large bright clusters. [Figure 3F] Representative photographs of cytotoxicity assays after 24 hours of co-incubation according to Example 3. Figure 3F shows a control using non-transduced CAR T cells from a healthy donor. [Figure 4A]

[0023] Figure 4A is a graph of a CAR T cell killing assay performed according to Example 3. Figure 4A shows the results from co-incubating OCI-LY1_wt cells (OCI wt) with CAR T cells from different healthy donors as indicated. As can be seen from Figure 4A, OCI-LY1_wt cells were killed by anti-CD19 CAR T cells (CARHDCD19) but remained unaffected by CAR T cells comprising humanized anti-IGLV3-21R110 CAR (CARHDh2). Comparing Figures 4A and 4B readily reveals that, unlike anti-CD19 CAR T cells, anti-IGLV3-21R110 CAR T cells selectively kill the IGLV3-21R110-positive cell line OCI-LY1_R110. [Figure 4B]

[0023] Figure 4A shows graphs of CAR T cell killing assays performed according to Example 3. Figure 4A shows results from co-incubation of OCI-LY1_wt cells (OCI wt) with CAR T cells from different healthy donors as indicated. Figure 4B shows the same set of assays for OCI-LY1_R110 cells. As can be seen from Figure 4A, OCI-LY1_wt cells were killed by anti-CD19 CAR T cells (CARHDCD19) but remained unaffected by CAR T cells containing humanized anti-IGLV3-21R110CAR (CARHDh2). Comparing Figures 4A and 4B readily reveals that, unlike anti-CD19 CAR T cells, anti-IGLV3-21R110 CAR T cells selectively kill the IGLV3-21R110-positive cell line OCI-LY1_R110. [Figure 5A] 5A is a graphical representation of cytokine quantification in 24-hour co-culture supernatants from the killing assay shown in Figure 4. As can be seen in Figure 5A, supernatants that demonstrated cell killing by CAR T cells had elevated levels of IFN-γ and IL-6 secretion. [Figure 5B] 5B is a graphical representation of cytokine quantification in 24-hour co-culture supernatants from the killing assay shown in Figure 4. As can be seen in Figure 5B, supernatants that demonstrated cell killing by CAR T cells had elevated levels of IFN-γ and IL-6 secretion. [Figure 6A]

[0033] Figure 6A is a graphical representation of killing calculated from a CAR T cell killing assay after 24 hours of co-incubation according to Example 3. Figure 6A shows the killing activity of primary CLL cells from IGLV3-21R110-negative CLL cases (CLL426 and CLL427) by healthy donor-derived anti-IGLV3-21R110 CAR T cells (CARHDh2), anti-CD19 CAR T cells (CARHDCD19), and anti-TSHR control CAR T cells (CARHDTSHR) compared to untransduced cells (CARHDUTD), as indicated. As shown in a comparison of Figures 6A and 6B, CD19-directed CAR T cells killed primary CLL cells from both IGLV3-21R110-positive and -negative CLL cases, whereas anti-IGLV3-21R110 CAR T cells selectively recognized and killed IGLV3-21R110-positive CLL cells. [Figure 6B]

[0033] Figure 6B is a graphical representation of killing calculated from a CAR T cell killing assay after 24 hours of co-incubation according to Example 3. Figure 6B shows the same analysis as shown in Figure 6A for killing of primary CLL cells from IGLV3-21R110-positive CLL cases (CLL438, CLL442). As shown in a comparison of Figures 6A and 6B, CD19-directed CAR T cells killed primary CLL cells from both IGLV3-21R110-positive and -negative CLL cases, whereas anti-IGLV3-21R110 CAR T cells selectively recognized and killed IGLV3-21R110-positive CLL cells. [Figure 7] Figure 1 is a graph of a CAR T cell killing assay quantifying polyclonal B cell killing mediated by healthy donor-derived CAR T cells compared to untransduced cells (CARHDUTD). The assay was performed according to Example 3. As can be seen from the graph, polyclonal healthy B cells were killed by anti-CD19 CAR T cells (CARHDCD19) but remained unaffected by anti-IGLV3-21R110 CAR T cells, demonstrating that epitope-specific targeting spares normal B cells. [Figure 8A]8A-8C are graphical representations of cytokine quantification in 24-hour co-culture supernatants from the killing assays shown in Figures 6 and 7. As can be seen in Figure 8A, supernatants that demonstrated cell killing by CAR T cells had elevated levels of IFN-γ and IL-6 secretion. [Figure 8B] 8B is a graphical representation of cytokine quantification in 24-hour co-culture supernatants from the killing assays shown in Figures 6 and 7. As can be seen in Figure 8B, supernatants that demonstrated cell killing by CAR T cells had elevated levels of IFN-γ and IL-6 secretion. [Figure 9A]

[0033] Figure 9A is a graphical representation of killing calculated from a CAR T cell killing assay after 24 hours of co-incubation according to Example 3. Figure 9A shows quantification of OCI-Ly1_wt (OCI wt) killing. Assays were performed on two CLL patients who served as T cell donors, one with active CLL (CLL433) and one with CLL in remission (CLL453). Comparison of Figures 9A and 9B clearly reveals that patient-derived anti-IGLV3-21R110 CAR T cells exhibit selective killing of Oci-Ly1-R110 (OCIR110), while anti-CD19 CAR T cells from the same patient cause killing regardless of the neoepitope. [Figure 9B]

[0033] Figure 9A and 9B are graphical representations of killing calculated from a CAR T cell killing assay after 24 hours of co-incubation according to Example 3. Figure 9B shows quantification of OCI-Ly1_R110 (OCIR110) killing mediated by CLL patient-derived CAR T cells containing an anti-IGLV3-21R110 CAR (CARCLLh2) or an anti-CD19 CAR (CARCLLCD19) compared to untransduced cells (CARCLLUTD). Assays were performed on two CLL patients who served as T cell donors, one with active CLL (CLL433) and one with CLL in remission (CLL453). Comparison of Figures 9A and 9B clearly reveals that patient-derived anti-IGLV3-21R110 CAR T cells exhibit selective killing of Oci-Ly1-R110 (OCIR110), while anti-CD19 CAR T cells from the same patient cause killing regardless of the neoepitope. [Figure 10]Figure 1 shows the determination of subtherapeutic doses of ibrutinib. The graph shows the titration of ibrutinib to determine toxicity in the OCI-Ly1 cell model when serial dilutions (11 steps, ranging from 0.01 to 20 μM) of ibrutinib were administered to OCI-Ly1 wt or OCI-Ly1_R110 cells for 24 hours. Percent cell proliferation was plotted as the total number of ibrutinib-treated cells relative to the total number of untreated cells. This analysis revealed that 0.08 μM ibrutinib was the highest dose that did not cause a decrease in target cell viability. Therefore, this dose was selected for further testing of CAR T efficacy. [Figure 11] 1 is a kill graph calculated from a CAR T cell killing assay according to Example 4 showing the effect of subtherapeutic doses of the BTK inhibitor ibrutinib. The graph shows the addition of all cells and ibrutinib. The graph shows time-dependent monitoring of the exclusive killing of an IGLV3-21R110-expressing cell line (OCIR110) by anti-IGLV3-21R110 CAR T cells derived from a healthy donor (CARHDh2) and anti-IGLV3-21R110 CAR T cells derived from the blood of a CLL patient (CLL425, CARCLLh2). Superior killing was observed with anti-IGLV3-21R110 CAR-based CAR T cells derived from healthy donor T cells, while CAR T cells derived from CLL patients still demonstrated efficient killing capabilities. Furthermore, healthy donor polyclonal B cells (BHD) were unaffected by these CAR T cells, demonstrating that healthy donor and CLL patient CAR T cells can selectively kill IGLV3-21R110-positive cells while sparing healthy B cells. Furthermore, as shown by comparing cytotoxicity with and without ibrutinib, the killing ability of healthy donor-derived CAR T cells was unaltered by the presence of subtherapeutic doses of ibrutinib throughout the 16-hour monitoring period. While the killing ability of CLL patient-derived CAR T cells decreased over time in the absence of ibrutinib, the presence of ibrutinib elevated the killing ability of CLL patient-derived cells to the level of healthy donor CAR T cells throughout the monitoring period. [Figure 12]Figure 12 is a graphical representation of the cytokine IFN-γ secretion by CAR T cells in the 16 hour co-culture supernatants from the killing assay shown in Figure 11. As can be seen in Figure 12, elevated levels of IFNg were measurable in each of the supernatants, which indicated cell killing by CAR T cells. [Figure 13] Schematic diagram of the BCR-BCR homotypic interaction of the IGLV3-21R110 light chain (described by Minici et al.). Two adjacent BCRs are shown with antigen-binding subunits comprising heavy (HC) and light (LC) chains, transmembrane domains (TM), and signaling subunits (SU) composed of disulfide-linked heterodimers of Igα and Igβ proteins (CD79a / CD79b). A mutant arginine (R) at position 110 of one BCR interacts with a germline-encoded aspartic acid (D) at position 50 of the adjacent BCR. Further interaction between the two BCRs is mediated by the germline-encoded amino acid residues lysine (K) at position 16 and aspartic acid (D) at position 52. [Figure 14A] Figure 14A is a schematic diagram of IGLV3-21R110. Figure 14A: Illustration of IGLV3-21R110 (SEQ ID NO: 41) in single letter code. Amino acid residues involved in BCR-BCR homotypic interactions according to Minici et al. are shown in bold. [Figure 14B] Schematic diagram of IGLV3-21R110. Figure 14B: Line 1: Amino acid positions of IGLV3-21R110. Line 2: Amino acid residues involved in BCR-BCR homotypic interactions according to Minici et al. Line 3: YDSD motif of IGLV3-21R110. Amino acids are shown in three-letter code. [Figure 15] FIG. 1 shows the sequences of SEQ ID NOs: 1 to 55.

[0134] The present invention is further illustrated by the following examples, which are presented solely to illustrate the invention with reference to specific embodiments. These exemplifications illustrate certain specific aspects of the invention, but do not represent limitations on or limit the scope of the disclosed invention.

[0135] All examples were carried out using standard techniques known and routine to those skilled in the art, unless otherwise specified. Routine molecular biology techniques in the following examples can be performed as described in standard laboratory manuals, such as Sambrook et al., 1989 supra.

[0136] Preferred embodiments of the present invention are as follows: Example Example 1 Mouse anti-IGLV3-21 R110 Antibody-derived humanized scFv and humanized anti-IGLV3-21 R110 CAR design 1. Antibodies 1.1. Mouse monoclonal antibody mAb01-01 The mouse monoclonal antibody mAb01-01 is IGLV3-21 R110 This antibody was developed by immunizing mice with a soluble form of the BCR (see SEQ ID NOs: 42 and 43) carrying the BCR, in combination with the selection of suitable antibodies using a cell line in which an intact and functional BCR is displayed in a membrane-bound manner.

[0137] First, it was necessary to obtain a soluble form of BCR in the form of IgG1 for immunization of mice. Therefore, a DNA segment encoding IGHV3-21 as an exemplary variable heavy chain (VH) and IGLV3-21 were prepared. R110Complete light chain (LC) DNA covering the VH and LC regions was synthesized by a contract manufacturer using standard procedures. These were then fused to mouse IgG1 constant segments by polymerase chain reaction (PCR) and cloned into a cytomegalovirus (CMV) vector. For expression of these IgG1s (SEQ ID NO: 42 for VH and SEQ ID NO: 43 for LC), a human cell expression system based on HEK293T cells was used, as previously described, for example, in Rekombinante Antikoerper, Lehrbuch und Kompendium für Studium und Praxis, 2. Auflage, Springer Verlag 2019. A polyethyleneimine (PEI)-based protocol was used for transfection. After several passages, the supernatants were pooled, and the medium contained in the combined cell supernatants was purified using a protein G column. The purity and quality of the soluble IgG1 were determined by Western blot.

[0138] Mice were then immunized with a recombinantly produced soluble form of BCR (see SEQ ID NOs: 42 and 43).

[0139] Immune cells with the desired specificity could then be obtained from these mice and transformed into hybridoma cells by cell fusion. Monoclonal antibodies were produced using standard procedures in mice and subsequent generation of hybridoma cells.

[0140] Screening for positive clones was not performed by conventional enzyme-linked immunosorbent assay (ELISA). Because the target structure is a membrane-bound receptor, it is crucial to verify the binding of potential antibodies in a cell line, i.e., while maintaining a cellular physiological state specific to this cell type. First, we examined binding events in pooled supernatants using fluorescence-activated cell sorting (FACS) analysis. To this end, we expressed different BCR mutants on the surface of a triple knockout (TKO) cell line that is unable to express the BCR itself.

[0141] The starting point for generating TKO cells is transgenic mice in which the Lambda5, RAG1 or RAG2, and SLP65 genes are knocked out, respectively (Duehren von Minden et al., 2012, Nature 489, pp. 309-313). The combination of knockout of RAG2 or RAG1 and Lambda5 results in a blockade of the pro-B to pre-B cell transition, classically characterized by the onset of heavy chain VDJ segment rearrangement. These cells are therefore pro- / pre-B cells. BCR activity can be measured by reconstitution with inducible SLP65. The generation of such mice is known to experts and represents the state of the art. To obtain the cells, mice were sacrificed and femoral bone marrow was extracted from them. The resulting cells were then cultured under conditions that promote the survival of pro- / pre-B cells (37°C, 7.5% CO2, Iscove's medium, 10% FCS, P / S, mouse IL-7). After several passages, FACS sorting is performed for control purposes to select pro / pre-B cells, which are then returned to culture. The markers used for this purpose are known to the expert.

[0142] To reconstitute the "desired BCR," the corresponding VH-encoding sequence was fused to a human IgM constant segment by polymerase chain reaction (PCR), and the heavy chain (HC) and light chain (LC) were cloned into their respective expression vectors, each containing a CMV promoter. These were then introduced into a packaging cell line (Phoenix cell line) by lipofection. After 36 hours of incubation, the viral supernatant was removed and used for spinfection of TKO cells. BCR expression was determined by FACS using anti-IgM and anti-LC antibodies. For this purpose, several cells were harvested and stained with 5 μl of each antibody in a total volume of 100 μl in PBS. Both the supernatant extraction and TKO spinfection are well-known procedures and familiar to experts. Knockout of RAG2 or RAG1 and Lambda5 ensured that only the "desired BCR" was expressed on the surface.

[0143] In this way, we generated two different BCR-expressing TKO cell lines, one of which expresses membrane-bound IGHV3-21 / IGLV3-21. R110 To generate a second BCR-expressing TKO cell line, IGLV3-21 R110 The codon for arginine at position 110 of the DNA encoding IGLV3-21 (IGLV3-21) was reverted to the germline sequence by known site-directed mutagenesis techniques (see, e.g., Sambrook et al., 1989 supra). The resulting TKO cells were designated IGLV3-21, which has a glycine at amino acid position 110 (IGLV3-21). G110 To generate a third control TKO cell line that does not express a BCR on its surface, spinfection with an empty expression vector was performed. Reconstituting the cells with inducible SLP65 allowed us to characterize the function of the expressed BCR, thereby enabling the expression of IGHV3-21 / IGLV3-21 on the surface. R110 The autonomously active state of the BCR could be verified before selection. The method chosen here was to use FACS analysis to detect Ca2+ receptors such as Indo-1. 2+ The first step is the measurement of Ca flux after induction of SLP65 using a Ca-dependent dye. These methods are known to experts (see M. Duehren-von Minden et al.; Nature 2012). By "targeting" these cells, FACS is currently being used to analyze IGLV3-21 R110 This method has been used to identify antibodies that specifically bind to BCRs carrying IGHV3-21 / IGLV3-21. The first step was to identify supernatants to which the antibodies showed binding. In this first selection round, supernatants from several clones were combined and examined for binding profiles. R110A positive binding profile is obtained when specific binding to the -BCR is demonstrated. Groups showing such profiles were isolated, and the binding profiles of individual clones were characterized again in a second selection round. The binding of the monoclonal antibody was verified using a FACS binding assay using a fluorescently labeled anti-mouse IgG antibody. This approach allowed the isolation of monoclonal antibody mAb01-01, which binds to wild-type IGLV3-21, which has a germline-encoded glycine at amino acid position 110. G110 Human IGLV3-21 without any detectable binding to the mutant R110 - It showed high specificity and cross-reactivity to BCR.

[0144] Antibody mAb01-01 was sequenced. The CDRs were identified based on the IMGT numbering amino acid annotation. The sequences corresponding to the light chain CDRs L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NOs: 1, 2, and 3, and the sequences corresponding to the heavy chain CDRs H-CDR1, H-CDR2, and H-CDR3 are shown in SEQ ID NOs: 4, 5, and 6. The amino acid sequences of the mouse variable light and heavy chains were identified as SEQ ID NO: 44 for VL and SEQ ID NO: 45 for VH.

[0145] 1.2. Humanized anti-IGLV3-21 R110 antibody 1.2.1 Humanization of mAB01-01 Humanization of the VL and VH regions was performed by in silico grafting of murine CDRs onto mature human frameworks of the variable light and heavy chain regions using standard CDR-grafting techniques by Fusion Antibodies Plc, Belfast, Northern Ireland. Multiple human framework sequences were identified for use as "acceptor" frameworks for the mAB01-01 CDR sequences. All of these acceptor sequences were derived from mature human IgG from human sources. As a result, the humanized sequences are expected to be non-immunogenic and retain the canonical structure of the CDR loops.

[0146] The human germline gene V region closest to the mouse VH domain was Homo sapiens IGHV4-59. The human germline gene V region closest to the mouse VL domain was Homo sapiens IGKV1-9. New variants were designed based on previous variants that demonstrated binding. Germline variants similar to the parent antibody but with more differentiated frameworks were also selected, CDR-grafted, and backmutated to maintain binding. Key residues important for the VL / VH interface and canonical loop structure were maintained as much as possible in the humanized variants using the CDRx platform (Fusion Antibodies Plc, Belfast, Northern Ireland).

[0147] The humanized variants were checked to determine whether they were humanized according to the WHO definition of a humanized antibody (see also World Health Organization (WHO) International Nonproprietary Names (INN) for biological and biotechnological substances (a review) 2014): the variable regions of the humanized chains have V-region amino acid sequences (typically derived from V-region gene segments after somatic hypermutation) that, when analyzed as a whole, are closer to the V-region germline sequences of humans (i.e., before somatic hypermutation) than to other species, as assessed using the Immunogenetics Information System® (IMGT®) DomainGapAlign tool (Ehrenmann F, Kaas Q and Lefranc MP 2010 supra).

[0148] Table 1 summarizes the best aligned V genes and alleles, including species, IMGT V gene and allele names, and domain labels, along with the identity percentage resulting in the WHO INN prediction for the humanized variant, and the overlap (number of aligned amino acids assigned to the V region) between the humanized variant and the IMGT amino acid reference sequence.

[0149] [Table 1]

[0150] 1.2.2 Humanized anti-IGLV3-21 R110 Antibody expression The amino acid sequences of the humanized variants generated by the fusion antibodies were then converted to nucleotide sequences using Geneiouse software (Geneious Prime 2, Auckland, New Zealand). Humanized heavy and light chains were generated by PCR by fusing the VH sequence with a human IgG1 isotype constant domain sequence (SEQ ID NO: 50 for IgG1 constant nucleotides) and the VL sequence with a human IgK isotype constant domain (SEQ ID NO: 51 for IgK constant nucleotides). The antibody gene sequences were transiently expressed in Chinese hamster ovary cells (CHO). The resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. The humanized antibody was purified from the cell culture supernatant by affinity chromatography. The purity of the antibody was determined to be greater than 95% by reducing and denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein content and concentration of the antibody were analyzed by size-exclusion chromatography (SEC) in PBS buffer. All steps were performed using state-of-the-art equipment and techniques.

[0151] This approach resulted in eight humanized antibodies: HC6-LC6, HC7-LC6, HC6-LC7, HC7-LC7, HC6-LC8, HC7-LC8, HC6-LC9, and HC7-LC9.

[0152] 1.2.3 Humanized anti-IGLV3-21 R110 Antibody binding affinity Eight humanized antibodies, HC6-LC6, HC7-LC6, HC6-LC7, HC7-LC7, HC6-LC8, HC7-LC8, HC6-LC9, and HC7-LC9, were expressed, followed by the soluble form of IGLV3-21. R110 BCR binding studies have shown that non-humanized antibodies (non-theof) have a binding activity of approximately 3.10 -10 The values ​​of >M confirmed the high affinity of these antibodies.

[0153] In short, IGLV3-21 R110 To define the binding affinity of the antibody to the B cell receptor carrying the IGLV3-21 antibody, a soluble recombinant version of the BCR (170.5 kDa; sequences according to SEQ ID NO: 52 for the HC and SEQ ID NO: 53 for the LC) was produced as a monomeric human IgM by transient expression in the 293-HEK cell line using the protocol described in section 1.1 of this example and bound to immobilized anti-IGLV3-21 antibody. R110 Binding to the antibodies was monitored by biolayer interferometry (BLI) on a Fortebio Octet instrument (Satorius).

[0154] The kinetic assay first detects humanized anti-IGLV3-21 antibody via an indirect capture reagent, an anti-human IgG Fc antibody. R110 Anti-IGLV3-21 antibodies were immobilized on a biosensor. R110 The antibody was loaded at a concentration of 0.01875 μg / ml, and 0.30–0.34 nm of anti-IGLV3-21 R110 To determine the antibody capture level, a 9 nM BCR fragment solution was prepared in running buffer (PBS, 0.02% Tween 20, 0.1% BSA, 0.05% sodium citrate) and serially diluted 1:3 to obtain seven concentrations ranging from 9 to 0.012 nM (9 nM, 3 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, and 0.012 nM). Anti-IGLV3-21 was then used. R110The antibody capture biosensor was immersed in wells containing different concentrations of soluble BCR fragments for 900 seconds (association step), followed by a dissociation step in running buffer for 1200 seconds. The steps were performed at a constant shaking speed of 1000 rpm. All reagents were used as described by the manufacturer. Captured anti-IGLV3-21 R110 Sensorgrams were generated after double-reference correction (buffer and blank sensor) to correct for both the spontaneous dissociation of the antibody and the nonspecific binding of soluble BCR fragments to the sensor surface. The dissociation rate constant (K D ) is the association (k) obtained by fitting the sensorgram with a first-order 1:1 binding model using Fortebio data analysis software (Satorius). a ) and dissociation rate (k d ) calculated based on the ratio of constants.

[0155] [Table 2]

[0156] 2. Humanized anti-IGLV3-21 R110 CAR 2.1. Humanized anti-IGLV3-21 R110 CAR generation Subsequently, a structural model of a humanized single-chain variable fragment (scFv) containing the amino acid sequences of the humanized LC6 and HC7 variants fused with a linker (Li, SEQ ID NO: 46) was predicted (Schroedinger Suit, Schroedinger Inc., New York, USA). The resulting sequence requirements were used as a template for the design of the remaining single-chain fragments.

[0157] The amino acid sequence of the humanized scFv was converted to a nucleotide sequence using Geneiouse software (Geneious Prime 2, Auckland, New Zealand), and the scFv was converted to a humanized anti-IGLV3-21 R110 CARs were designed using two different CAR constructs: CAR construct 1 contains a human IgG4 hinge-derived spacer (UniProtKB P01861 IGHG4_HUMAN). The costimulatory domain is derived from the CD28 transmembrane domain (UniProtKB P10747 CD28_HUMAN) and human receptor 4-1BB (UniProtKB Q07011 TNR9_HUMAN). CD3ζ (UniProtKB P20963 CD3Z_HUMAN) is used as the signaling domain.

[0158] CAR construct 2 differs from CAR construct 1 in that it contains an additional spacer sequence derived from the CH2-CH3-domain of IgG4 (UniProtKB P01861 IGHG4_HUMAN) between the IgG4 hinge and the CD28 sequence.

[0159] The resulting humanized anti-IGLV3-21 R110 The sequences of the antigen-binding domains of the CARs are shown in Table 3.

[0160] [Table 3]

[0161] 2.2.T cell epitope screening Presentation of peptide sequences in the groove of MHC class II molecules leads to CD4+ T cell activation and immunogenic responses. To reduce this response, therapeutic proteins can be designed to reduce binding affinity to MHC class II molecules and thereby avoid incorporating T cell epitopes that may activate T cells.

[0162] anti-IGLV3-21 R110To demonstrate that the antigen-binding domain of the CAR exhibits reduced immunogenicity compared to mouse-derived sequences, the humanized VL and VH protein sequences of the binding domain and the sequence of the mouse antibody mAB01-01 were screened for MHC class II-binding peptides. Screening was performed using SMM-align, a method known in the art (Wang P, Sidney J, Kim Y, et al. 2010. Peptide binding predictions for HLA DR, DP, and DQ molecules. BMC Bioinformatics. 11:568; Nielsen M, Lundegaard C, Lund 0. 2007. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinformatics. 8:238). The following eight alleles, representing over 99% of the world's population, were used as the standard allele set for predicting MHC class II epitopes: DRB1 * 01:01;DRB1 * 03:01;DRB1 * 04:01;DRB1 * 07:01;DRB1 * 08:02;DRB1 * 11:01;DRB1 * 13:02;DRB1 *15:01(Gonzalez-Galarza FF, Christmas S, Middleton D and Jones AR 2011. Allele frequency net: a database and online repository for immune gene frequencies in worldwide populations. Nucleic Acid Research, 39, D913-D919, Greenbaum J, Sidney J, Chung J, et al. 2011 Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes. Immunogenetics 63(6):325-35).

[0163] Tables 4 and 5 show the results of the VL and VH screening, with the high affinity T cell epitope core in bold (IC50<50 nM).

[0164] The results of the VL screening are shown in Table 4. The human germline sequence IGKV1-9 was also analyzed for comparison, and any potential T cell epitopes present in the germline sequence and matched in the humanized variants are italicized.

[0165] [Table 4]

[0166] The results of the VH screening are shown in Table 5. The human germline sequence IGHV4-59 was also analyzed for comparison, and any potential T cell epitopes present in the germline sequence and matched in the humanized variants are italicized.

[0167] [Table 5]

[0168] As can be readily seen from Tables 4 and 5, the murine VH contains a T cell epitope within the framework 1 region of the sequence that is not present in the humanized variants (HC6, HC7).

[0169] Example 2 Generation of primary human T cells expressing CAR Immune cell specificity and reactivity with IGLV3-21 R110 Humanized anti-IGLV3-21 redirecting positive BCR R110 To explore the potential of CAR, humanized anti-IGLV3-21 R110 T cells expressing CAR H1 and H2 were generated by viral vector transduction of T cells using techniques known in the art and previously described (e.g., Yang S, Zhou X, Li R, Fu X, Sun P. Optimized Pei-Based Transfection Method for Transient Transfection and Lentiviral Production. Curr Protoc Chem Biol (2017) 9(3): 147-57. Epub 2017 / 09 / 15. doi: 10.1002 / cpch.25). To test T cells in different states, healthy humans and CLL patients with active CLL (CLL425; CLL433) and CLL in remission (CLL453) served as T cell donors.

[0170] Preparation of CAR gene delivery vehicle particles (lentiviral vector particles) For expression of scFv in CAR construct 1, vector pJ2459_19loBB (SEQ ID NO: 48) was used, and for expression of scFv in CAR construct 2, vector pJ2487_19loBB (SEQ ID NO: 49) was used. Humanized anti-IGLV3-21 R110 Both vectors for expression of CAR are shown in FIG.

[0171] The scFv sequence was cloned into two CAR vectors, pJ2487_19loBB and pJ2459_19loBB, using the Nhel (New England Biolabs, catalog number R3131M) and RsrII (New England Biolabs, catalog number R0501S) restriction sites using the In-Fusion® Cloning Kit (Takara Bio Inc., catalog number 639650) according to the manufacturer's instructions.

[0172] Lentiviral vector particles were produced in HEK293T cells (DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Cat. No. ACC 635). 5 × 10 6 25cm of HEK293T cells 2Cells were seeded in culture flasks in DMEM GlutaMAX™ (Thermo Fisher Scientific, Cat. No. 31966021) supplemented with 10% (v / v) FBS (Life Technologies, Cat. No. 10500064) and 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher Scientific, Cat. No. 15140122) and incubated at 37°C in a humidified atmosphere containing 5% CO. After 18–24 h, the medium was replaced with 10 mL of fresh medium containing 25 μM chloroquine (Sigma, Cat. No. C6628). Fifteen micrograms of CAR construct vector was mixed with 10 μg of gag / pol plasmid pMDLg / pRRE (Addgene, Catalog No. 12251), 5 μg of rev plasmid pRSV-Rev (Addgene, Catalog No. 12253), and 2 μg of envelope plasmid pCMV-VSV-G (Addgene, Catalog No. 8454) in 500 μL of 250 mM CaCl2 (Sigma, Catalog No. C1016). 500 μL of 2x HEPES-buffered saline (Thermo Fisher, Catalog No. 15488749) was added to the DNA mixture, incubated at room temperature for 20 minutes, and added to the cells. Cell culture media was harvested after 24, 48, and 72 hours, filtered through a 0.45 μm syringe filter (Sarstedt, Catalog No. 83.1826), and stored at -80°C until transduction.

[0173] Isolation and transduction of T cells Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor or CLL patient blood collected in K3 ethylenediaminetetraacetic acid (EDTA) S-Monovette (EDTA tubes, Sarstedt AG, catalog number 02.1066.001) EDTA tubes. Whole blood was layered onto lymphocyte separation medium (Anprotec, catalog number AC-AF-0018) and centrifuged at 500 × g for 30 minutes without rest. PBMCs were collected from the interphase and washed with phosphate-buffered saline (PBS). For CLL samples only, B cells were depleted from PBMCs using the human B Cell Isolation Kit II (Miltenyi, catalog number 130-091-151) on a Miltenyi autoMACS® instrument according to the manufacturer's instructions. T cells were isolated based on negative selection using the Pan T Cell Isolation Kit (Miltenyi, Cat. No. 130-096-535) on a Miltenyi autoMACS® instrument according to the manufacturer's instructions.

[0174] All T cells were grown at 37°C in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium with GlutaMAX™ Supplement (Thermo Fisher, Catalog No. 61870044) (CTL medium) supplemented with 10% (v / v) FBS (Life Technologies, Catalog No. 10500064), 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher, Catalog No. 15140122), 50 μM β-mercaptoethanol (Thermo Fisher, Catalog No. 31350010), and supplemented with 50 U / mL IL-2 (StemCell, Catalog No. 78036.1) every 48 hours. One million (1 Mio) T cells were plated in 2 mL of CTL medium per well of a 24-well plate with washed CD3 / CD28 beads (ImmunoCult™ Human CD3 / CD28 T Cell Activator, StemCell, catalog no. 10971) at a 1:1 bead:T cell ratio and 50 U / mL IL-2 (StemCell, catalog no. 78036.1). After 24 hours, 1.3 mL of medium per well was removed, 5 μg / mL polybrene (Merck, catalog no. TR-1003-G) and lentiviral particles were added, and the cells were centrifuged at 800 g and 32°C for 45 minutes without stopping. Cells were incubated at 37°C for 4 hours, and fresh CTL medium was added. Half of the medium was replaced every two days from the following day, and fresh IL-2 was added. After 6 days, the CD3 / CD28 beads were removed. Cells were expanded for up to 21 days. Transduction efficiency was confirmed by flow cytometry using the anti-EGFR antibody cetuximab on a BD FACSCalibur™ instrument (E cells in the fluorescent channel FL1 above the threshold of the unstained control were considered successfully transduced). Transduction efficiencies of over 80% were observed.

[0175] Generated anti-IGLV3-21 R110 CAR-T cells are shown in Table 6.

[0176] [Table 6]

[0177] Example 3 Humanized anti-IGLV3-21 R110 In vitro cytotoxicity assay using CAR-T cells A. Target and Effector Cell Preparation a) Target cells: CAR efficacy was tested using OCI-LY1 wt, OCI-LY1 R110, primary CLL PBMCs or healthy B cells.

[0178] a.1) OCI-LY1 wt cell line (OCI wt) The OCI-LY1 (wild type) wt cell line, derived from the bone marrow of a 44-year-old man with B-cell non-Hodgkin's lymphoma, was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, catalogue number ACC722).

[0179] a.2) OCI-LY1 R110 cell line (OCIR110) IGLV3-21 as part of the BCR in which the R110 point mutation was induced from OCI-LY1 wt cells R110 We generated a neoepitope (i.e., cancer-specific peptide)-modified cell line, OCI-LY1 R110, expressing a light chain. First, OCI-LY1 wt cells were cultured in RPMI 1640 medium supplemented with 20% (v / v) FBS (Life Technologies, Cat. No. 10500064) and 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher, Cat. No. 15140122), GlutaMAX™ Supplement (Thermo Fisher, Cat. No. 61870044), in a humidified atmosphere containing 5% CO2 at 37°C. IGLV3-21 as part of the hybrid BCR R110For ectopic expression of the light chain, the coding sequence (SEQ ID NO: 47) was cloned into the lentiviral Gene Ontology (LeGO) vector LeGO-iC2-Puro+ (Addgene, Cat. No. 27345) via AsiSI (New England Biolabs, Cat. No. R0630S) / EcoRI (New England Biolabs, Cat. No. R3101L). Lentiviral particles were produced from HEK293T cells (DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Cat. No. ACC 635) according to the manufacturer's protocol, and OCI-LY1 wt cells were transduced with the viral particles. As already pointed out in connection with Example 2, the work of producing lentiviral particles and the transduction of cells are both widely known procedures and known to experts (see, e.g., Yang S et al. (2017) supra; Weber K, Bartsch U, Stocking C, Fehse B. A Multicolor Panel of Novel Lentiviral "Gene Ontology" (Lego) Vectors for Functional Gene Analysis. Molecular Therapy (2008) 16(4): 698-706). R110 Light chain expression was measured using APC-anti-IGLV3-21 R110 Confirmation was performed by flow cytometry using an antibody (AVA LifeScience, catalog number AVA-D01-APC / 01P0250).OCI-LY1 R110 cells were cultured as described for OCI-LY1 wt.

[0180] a.3) First generation CLL PBMC Primary CLL PBMCs were cultured from CLL patients with the R110 point mutation (CLL425; CLL438; CLL442) and from wt IGLV3-21 mice affected by different types of CLL. G110Blood samples were isolated from light chain-positive CLL patients (CLL399; CLL426; CLL427) and collected in EDTA tubes. Whole blood was layered onto lymphocyte separation medium (Anprotec, catalog number AC-AF-0018) and centrifuged at 500 x g for 30 minutes without rest. PBMCs were collected from the interphase and washed with phosphate-buffered saline (PBS).

[0181] a.4) Healthy B cells: Primary polyclonal B cells (B HD ) PBMCs were isolated from the blood of healthy donors and collected in EDTA tubes. Whole blood was layered onto lymphocyte separation medium (Anprotec, catalog number AC-AF-0018) and centrifuged continuously at 500 x g for 30 minutes. PBMCs were collected from the interphase and washed with phosphate-buffered saline (PBS). Primary polyclonal non-malignant B cells were isolated using the human B Cell Isolation Kit II (Miltenyi, catalog number 130-091-151) on a Miltenyi autoMACS® instrument according to the manufacturer's instructions.

[0182] b) Effector cells: b.1) Anti-IGLV3-21 R110 CAR T cells Humanized anti-IGLV3-21 R110 CAR T cells expressing CAR and prepared according to Example 2 were used, derived from healthy donors, CLL patients with active CLL (CLL425; CLL433) or CLL patients with CLL in remission (CLL453).

[0183] b.2) Non-transduced CAR T cells from healthy donors and CLL patients Non-transduced T cells from healthy donors (CARHDUTD, control) and CLL patients (eg, CLL433 CARCLLUTD; CLL453 CARCLLUTD) isolated according to Example 2 served to reduce background.

[0184] b.3) Anti-human thyrotropin receptor (TSHR) CAR T cells (CARHDTSHR) CAR T cells from healthy donors directed against the human thyrotropin receptor (HTRR) were used as a control. To generate anti-TSHR CAR T cells, the paratope sequence of an autoantibody targeting the human thyrotropin receptor (TSHR) was obtained from the literature (Rees Smith, B., Sanders, J., Evans, M., Tagami, T. & Furmaniak, J. TSH receptor - autoantibody interactions. Horm Metab Res 41, 448-455 (2009)), translated into scFv format, and cloned into the pJ2459 lentivector.

[0185] b.4) Anti-CD19 CAR T cells T cells from healthy donors (CARHDCD19) and CLL patients (e.g., CLL433 CARCLLCD19; CLL453 CARCLLCD19) were prepared by lentiviral transduction using the pJ2459-CD19-targeted CAR previously published by Hudecek, M., et al. (The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 3, 125-135 (2015)).

[0186] B. Cytotoxicity Assay 2 x 10 cells in a 96-well plate 4Target cells, seeded at 10 cells / well, were co-incubated with effector cells at an effector-to-target (E:T) ratio of 5:1 in complete CTL medium consisting of RPMI 1640 medium with GlutaMAX™ Supplement (Thermo Fisher, Cat. No. 61870044) supplemented with 10% (v / v) FBS (Life Technologies, Cat. No. 10500064), 1% (v / v) penicillin-streptomycin (10,000 U / mL) (Thermo Fisher, Cat. No. 15140122), 50 μM β-mercaptoethanol (Thermo Fisher, Cat. No. 31350010), and 50 U / mL IL-2 (StemCell, Cat. No. 78036.1). CAR T cell-mediated tumor cell killing was assessed using IncuCyte® Caspase-3 / 7 Green Reagent for Apoptosis (Sartorius, Cat. No. 4440) according to the manufacturer's instructions. Readings of dead (=green fluorescent) target cells were performed on an IncuCyte® S3 (Sartorius) after 2–16 or 4–36 hours. Killing graphs were calculated using IncuCyte® software (version 2022A). Total fluorescence for each well and time point was measured relative to control wells and recorded after specific time points or plotted over time. A combination of target cells and non-transduced T cells was used as a control to establish background. Additional control assays were performed with effector cells alone or target cells alone. All values ​​were obtained in triplicate.

[0187] As can be readily seen from Figure 3, which shows representative photographs of the cytotoxicity assay, and the killing graphs shown in Figures 4, 6, 7, and 9, unlike anti-CD19 CAR T cells, humanized anti-IGLV3-21 R110 CAR T cells carrying the scFv sequence were identified as IGLV3-21 R110 Selectively kills expressing cell lines and primary CLL cells.

[0188] Cytotoxicity assays also revealed that the killing ability of scFv H1 and scFv H2 was significantly higher in CAR construct 1 than in CAR construct 2, which may apparently be due to the shorter spacer between the binding domain and the transmembrane domain of CAR construct 1.

[0189] The time course monitoring shown in Figure 4 shows that anti-IGLV3-21 antibodies derived from a healthy human donor R110 IGLV3-21 with CAR T cells R110 Exclusive killing of expressing cell lines was demonstrated. R110 Negative cell lines were not affected by these CAR T cells.

[0190] Non-transduced control CAR T cells prepared from healthy donors or CLL patients were compared with IGLV3-21 R110 Neither the positive nor the negative cell lines were killed (Figures 4, 6, and 9). Similarly, both cell lines remained unaffected by anti-TSHR CAR T cells derived from healthy donors (Figures 4, 6, and 9).

[0191] Furthermore, cytotoxicity assays demonstrated that humanized anti-IGLV3-21 derived from a healthy human donor R110 Freshly prepared IGLV3-21 with CAR T cells carrying scFv sequences R110 Selective killing of CD19-positive CLL cells was demonstrated (Figure 6). Isolated primary polyclonal non-malignant B cells also remained unaffected, demonstrating that epitope-specific targeting spares normal B cells (Figure 7). In contrast, anti-CD19 CAR T cells inhibited the proliferation of IGLV3-21 R110 All B cells tested were killed similarly regardless of the presence of the epitope (eg, Figures 6 and 7).

[0192] IGLV3-21 R110To expand the study to a larger number of primary CLL target cells expressing the light chain, additional cytotoxicity assays were performed using thawed CLL samples. These experiments generally had higher background levels due to lower viability of primary CLL cells, but IGLV3-21 R110 Specific killing of positive CLL cells was confirmed.

[0193] Furthermore, as representatively shown in Figure 9 using killing graphs generated using CAR T cells derived from two CLL patients, one with active CLL and one with CLL in remission, CAR T cells derived from the blood of CLL patients demonstrated a significant reduction in the number of IGLV3-21 T cells, despite the well-recognized dysfunction of T cells in this disease. R110 It showed the ability to selectively kill positive cells.

[0194] Example 4 Humanized anti-IGLV3-21 in the presence of a BTK inhibitor R110 Comparative cytotoxicity assay using CAR-T cells Co-administration of CAR T cells with a BTK inhibitor, such as ibrutinib, a BTK / ITK inhibitor known to restore T cell functionality, may have an additive antitumor effect. To test this in the R110 CAR T setting, we first evaluated the toxicity of ibrutinib against OCI-Ly1 target cells. For this, we administered 2 × 10 4OCI-Ly1 wt and OCI-Ly1_R110 cells were seeded in 200 μl of culture medium supplemented with serially diluted ibrutinib (PCI-32765, Selleck Chemicals, catalog number S2680) ranging from 0.01 to 20 μM. Cells grown under the same conditions without ibrutinib served as a control. After 24 hours of incubation, viable (trypan blue-excluding) cells were counted using a Vi-CELL XR system (Beckman Coulter). Percent cell proliferation was then plotted as the total number of ibrutinib-treated cells relative to the total number of untreated cells (Figure 10). This analysis revealed that 0.08 μM ibrutinib was the highest dose that did not cause a decrease in target cell viability. Therefore, this dose was selected for further testing of CAR T efficacy.

[0195] Healthy donors and IGLV3-21 R110 Humanized anti-IGLV3-21 derived from a positive CLL patient (CLL425) R110 Comparative cytotoxicity assays using CAR-T cells were performed according to Example 3. For the IncuCyte® killing assay in the presence of a BTK inhibitor, 0.08 μM ibrutinib was added per well at the time of target and effector cell plating (day 0) for the duration of the assay (16 hours).

[0196] As shown in Figure 11, the addition of ibrutinib at subtherapeutic doses increased the killing capacity of CLL-derived CAR T cells to the level of CAR-T cells derived from healthy donors.

[0197] Example 5 Interferon-γ (IFNγ) profile The immune effector cytokine IFN-γ was confirmed during cytotoxicity assays. During co-culture of effector and target cells, IFN-γ concentrations in culture supernatants were measured after 8, 16, or 36 hours using the bead-based immunoassay technology LEGENDplex™ Human B Cell Panel Standard (BioLegend, catalog no. 740537) according to the manufacturer's protocol. 25 μL of supernatant was used per well. Fluorescence was measured using a BD FACSCelesta™ instrument. Values ​​below the detection limit were considered zero.

[0198] As shown in Figures 5A, 8A, and 12, in agreement with the results of the cytotoxicity assays, elevated IFNγ levels were measurable in the respective supernatants, indicating cell killing by the CAR T cells.

[0199] Example 6 Interleukin 6 (IL-6) profile The immune effector cytokine IL-6 was detected during cytotoxicity assays. IL-6 concentrations in culture supernatants from 8 to 36 hours after co-culture of effector and target cells were measured using the bead-based immunoassay technology LEGENDplex™ Human B Cell Panel Standard (BioLegend, catalog no. 740537) according to the manufacturer's protocol. 25 μL of supernatant was used per well. Fluorescence was measured using a BD FACSCelesta™ instrument. Values ​​below the detection limit were considered zero.

[0200] As shown in Figures 5B and 8B, elevated IL-6 levels were measurable in each supernatant, indicating cell killing by CAR T cells, consistent with the results of the cytotoxicity assay.

Claims

1. Humanized IGLV3-21 Ｒ１１０ A humanized chimeric antigen receptor (CAR) comprising a binding domain, a transmembrane domain, and a cytoplasmic domain, wherein the humanized IGLV3-21 Ｒ１１０ A CAR in which the binding domain includes a light chain variable region (VL) comprising a light chain complementarity determination region 1 (L-CDR1) having the amino acid sequence of SEQ ID NO: 1, a light chain complementarity determination region 2 (L-CDR2) having the amino acid sequence of SEQ ID NO: 2, and a light chain complementarity determination region 3 (L-CDR3) having the amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region (VH) comprising a heavy chain complementarity determination region 1 (H-CDR1) having the amino acid sequence of SEQ ID NO: 4, a heavy chain complementarity determination region 2 (H-CDR2) having the amino acid sequence of SEQ ID NO: 5, and a heavy chain complementarity determination region 3 (H-CDR3) having the amino acid sequence of SEQ ID NO:

6.

2. The CAR according to claim 1, characterized by any combination of VL having an amino acid sequence selected from the list consisting of SEQ ID NOs: 7, 8, 9, and 10, and VH having an amino acid sequence selected from the list consisting of SEQ ID NOs: 11 and 12.

3. The CAR according to claim 2, characterized by a VL having the amino acid sequence of SEQ ID NO: 7 and a VH having the amino acid sequence of SEQ ID NO: 11, or a VL having the amino acid sequence of SEQ ID NO: 7 and a VH having the amino acid sequence of SEQ ID NO:

12.

4. The CAR according to claim 3, characterized by having at least 70%, 75%, 80%, 85%, 90%, 95%, and 98% amino acid sequence identity with respect to the amino acid sequence of VL, and at least 70%, 75%, 80%, 85%, 90%, 95%, and 98% amino acid sequence identity with respect to the amino acid sequence of VH.

5. Humanized anti-IGLV3-21 Ｒ１１０ The CAR according to claim 1, wherein the binding domain is scFv.

6. The CAR according to claim 5, wherein the scFv has the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO:

20.

7. A polynucleotide encoding a CAR as described in claim 1.

8. A vector comprising the polynucleotide described in claim 7.

9. A cell expressing the CAR described in claim 1.

10. The cell according to claim 9, which is a T cell.

11. A pharmaceutical composition comprising the CAR according to claim 1, the polynucleotide according to claim 7, the vector according to claim 8, or the cell according to claim 9 or 10, and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition according to claim 11, used for the treatment of a disease.

13. The pharmaceutical composition according to claim 12, wherein the disease is chronic lymphocytic leukemia (CLL).

14. The pharmaceutical composition according to claim 12, which is administered in combination with an additional therapeutic agent.

15. The pharmaceutical composition according to claim 14, wherein the additional therapeutic agent is a Bruton's tyrosine kinase (BTK) inhibitor.

16. A kit comprising the pharmaceutical composition according to claim 11.

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