RU2830064C2 - CHIMERIC ANTIGEN RECEPTOR (CAR) WITH ANTIGEN-BINDING DOMAINS TO CONSTANT REGION OF β T-CELL RECEPTOR - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention relates to biotechnology and medicine, in particular to a method for diagnosing T-cell lymphoma or leukaemia in a subject, comprising a step of determining the percentage of T-cells that are TRBC1- or TRBC2-positive of the total number of T-cells in a sample, as well as to a method of selecting a suitable therapy for treating a subject having T-cell lymphoma or leukaemia, and a method of selecting a subject having T-cell lymphoma or leukaemia to receive a CAR-T cell therapy. Methods are carried out using an agent which contains an antigen-binding domain which selectively binds to a constant region of 1 TCR beta (TRBC1) or TRBC2.

EFFECT: invention provides a new method for targeted therapy of T-cell malignant neoplasms.

11 cl, 27 dwg, 5 tbl, 13 ex

Description

FIELD OF INVENTION

The present invention relates to cells and agents useful for the treatment of T-cell lymphoma or leukemia.

PRIOR ART

Lymphoid malignancies can be generally divided into those of either T-cell or B-cell origin. T-cell malignancies are a clinically and biologically heterogeneous group of disorders, collectively comprising 10–20% of non-Hodgkin lymphomas and 20% of acute leukemias. The most commonly identified histologic subtypes are peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITCL), and anaplastic large cell lymphoma (ALCL). Of all acute lymphoblastic leukemias (ALL), approximately 20% have a T-cell phenotype.

These diseases tend to behave aggressively compared to, for example, B-cell malignancies, with an estimated five-year survival of only 30%. In the case of T-cell lymphoma, they are associated with a high proportion of patients demonstrating disseminated disease, an unfavourable international prognostic indicator (IPI) value and a predominance of extranodal disease. Chemotherapy alone is generally ineffective, and less than 30% of patients are cured with current treatments.

Moreover, unlike B-cell malignancies, where immunotherapeutic agents such as the anti-CD20 monoclonal antibody rituximab have significantly improved outcome, there is currently no equally effective immunotherapeutic agent with minimal toxicity suitable for the treatment of T-cell malignancies. A significant challenge in developing immunotherapies for T-cell diseases is the significant overlap in the expression of clonal and normal T-cell markers, and the lack of a single antigen that can clearly identify clonal (malignant) cells.

A similar problem exists when targeting a pan-B-cell antigen for the treatment of B-cell malignancies. However, in this case, the concomitant depletion of the B-cell compartment results in relatively minor immunosuppression, which is well tolerated by most patients. Moreover, in therapeutic regimens that result in particularly prolonged depletion of the normal B-compartment, its loss can be largely counteracted by administration of mixed immunoglobulin. The situation is quite different when targeting T-cell malignancies. In this case, the concomitant depletion of the T-cell compartment results in severe immunosuppression and severe toxicity. Moreover, there is no satisfactory way to reduce the loss of the T-cell compartment.

Toxicity is partly illustrated by the clinical effects of the therapeutic monoclonal antibody alemtuzumab. This drug destroys cells that express CD52 and has some efficacy in T-cell malignancies. Its usefulness is severely limited by profound cellular immunodeficiency, mainly due to T-cell depletion, with markedly increased risk of infection.

There is thus a need for a new method for targeted therapy of T-cell malignancies that is not associated with the above disadvantages.

DESCRIPTION OF DRAWINGS

Figure 1: Diagram of the T cell receptor αβ/CD3 complex. The T cell receptor is formed from 6 different protein chains that must assemble in the endoplasmic reticulum to be expressed on the cell surface. The four proteins of the CD3 complex (CD3ζ, CD3γ, CD3ε, and CD3δ) comprise the T cell receptor (TCR). This TCR imbues the complex with specificity for a particular antigen and is composed of two chains: TCRα and TCRβ. Each TCR chain has a variable component located distal to the membrane and a constant component located proximal to the membrane. Almost all T cell lymphomas and many T cell leukemias express the TCR/CD3 complex.

Figure 2: Segregation of the T cell receptor β constant regions (TRBC)-1 and TRBC2 during T cell receptor rearrangement. Each TCR beta chain is formed by genomic recombination of distinct variable (V), diversity (D), junction (J), and constant (TRBC) beta regions. The human genome contains two highly similar and functionally equivalent TRBC loci, known as TRBC1 and TRBC2. During TCR gene rearrangement, the J region recombines with either TRBC1 or TRBC2. This rearrangement is permanent. T cells express many copies of a single TCR on their surface, so each T cell will express a TCR whose β chain constant region is encoded by either TRBC1 or TRBC2.

Figure 3: Alignment of human TRBC1 and TRBC2 at the amino acid level. The TCRβ constant chains encoded by TRBC1 and TRBC2 differ in only four amino acids: K/N at position 3 of TRBC, N/K at position 4 of TRBC; F/Y at position 36 of TRBC; V/E at position 135 of TRBC.

Figure 4: Unambiguous demonstration that the JOVI-1 antibody binds to TRBC1 and not TRBC2. We used cell genetic engineering to unambiguously demonstrate that the JOVI-1 monoclonal antibody recognizes the TRBC1 variant of the TCRβ constant chain. A tricistronic retroviral cassette was generated. It encodes both the TCRα and TCRβ chains of the human TCR that recognizes the minor histocompatibility antigen HA1, together with truncated human CD34 as a convenient marker gene. The HA1 TCR is natively TRBC2. A second retroviral cassette was generated that was identical to the first except that the four residues in the TCRβ constant region that distinguish TRBC1 from TRBC2 were replaced with residues encoded by TRBC1. Jurkat T cells knocked out for both TCRα and TCRβ chains were transduced with one of two vectors. The cells were stained with either pan-TCR/CD3 antibody or monoclonal JOVI-1 conjugated with anti-CD34 antibodies and analyzed by flow cytometry. The top row shows pan-TCR/CD3 antibody staining versus CD34 (a transduction marker), the bottom row shows JOVI-1 staining versus CD34. Transduced cells show similar TCR/CD3 staining, but only TRBC1-positive cells stain with JOVI-1. Thus, JOVI-1 is a specific antibody for TRBC1, and, moreover, it is possible to use the antibody to distinguish between TRBC1 and the two TCRs.

FIGURE 5: Monoclonal antibody JOVI-1 distinguishes TRBC1 from TRBC2 by recognizing residues 3 and 4 of TRBC. To determine precisely how JOVI-1 differentiates TRBC1 from TRBC2, the TRBC2 TCR HA1 construct described above was mutated to generate two TRBC1/2 fusions. An additional variant was generated such that only residues 3 and 4 of the TCRβ constant chain were changed from TRBC2 residues to TRBC1 residues. An additional variant was generated in which only residue 36 was changed from a TRBC2 residue to a TRBC1 residue. TCR knockout Jurkat T cells were transduced with these new constructs. Parental TRBC2- and TRBC1-transduced Jurkat cells described in Figure 4 served as controls. Jurkat T cells were stained with JOVI-1 and analyzed by flow cytometry. JOVI-1 staining overlapped with staining of TCR knockout and non-TCR-transduced Jurkat T cells. JOVI-1 stained Jurkat cells expressing TRBC1 TCR but not TRBC2 TCR. JOVI-1 stained TRBC1/hybrid 2, in which only TRBC residues 3 and 4 of TRBC were from TRBC1. JOVI-1 did not stain Jurkat T cells, in which only TRBC residue 36 was from TRBC1.

FIGURE 6: Example of TRBC1 T cell expression in a healthy donor. Healthy donor peripheral blood mononuclear cells were stained with antibodies against CD3, CD4, CD8, and JOVI-1 and analyzed by flow cytometry. The CD4+ and CD8+ T cell populations are shown in the top panel. Each of these populations gave a signal above the threshold, and forward scatter relative to JOVI-1 staining is shown on the y and x axes, respectively. These data show that both the CD4+ and CD8+ compartments contain cells that are TRBC1 positive and negative. These are representative data from a single donor.

Figure 7: TRBC1+ T cells in several healthy donors. Ten healthy donors were bled and peripheral blood mononuclear cells were stained as described above in Figure 4. Cumulative data of the proportion of TRBC1 T cells in both CD4 and CD8 compartments are shown in a bar graph along with median and range. All donors had both TRBC1+ and TRBC1- compartments. Median TRBC1+ cells in %=36%.

Figure 8: Cell lines derived from T-cell malignancies stained with JOVI-1. Several cell lines have been derived from T-cell malignancies. Many of these cell lines still express the TCR. We chose to examine Jurkat (a T-cell leukemia cell line), HPB-ALL (another T-cell leukemia cell line), and HD-Mar-2 (a T-cell lymphoma cell line). By staining these cell lines with a pan-TCR/CD3 antibody, we were able to demonstrate that all three lines express the TCR (left panels, staining overlaps with isotype control staining). Next, by staining with JOVI-1, we were able to determine that these T-cell lines are either TRBC1 negative or TRBC1 positive. Only Jurkat cells (TRBC1+), but not HPB-ALL or HD-Mar-2 cells (TRBC2+), stained with JOVI-1, confirming exclusive expression of either TRBC1 or TRBC2.

Figure 9: Selective lysis of TRBC1 T cells by the monoclonal antibody (mAb) JOVI-1. Wild-type Jurkat T cells (CD34-, TRBC1+) were mixed with TCRαβ knockout Jurkat T cells transduced with TRBC2 co-expressing the CD34 marker gene (CD34+TRBC2+). These cells were incubated with JOVI-1 alone or incubated with JOVI-1 and complement for 1 hour. Cells were washed and stained for CD34, annexin V, and 7-aminoactinomycin (7-AAD). Cells were analyzed by flow cytometry. CD34 expression in the live population defined by negative annexin-V and weak 7-AAD expression is shown below. (a) JOVI-1 alone; (b) JOVI-1 with complement. Selective lysis of TRBC1 T cells (CD34-) was observed.

Figure 10: Epstein-Barr virus (EBV)-specific polyclonal T cells can be divided into two approximately equal populations, TRBC1/2. Using established methods, we selectively expanded EBV-specific T cells from healthy donor peripheral blood. The resulting line was highly selective against autologous EBV-infected B cells (autolymphoblast cell lines, auto-LCLs), had no activity against allogeneic EBV-infected T cells (allo-LCLs), and no non-specific natural killer activity (as measured by the anti-K562 assay). This line is representative of donor immunity to EBV. (b) When stained with JOVI-1, these T cells are typed in equal proportions as TRBC1 and TRBC2.

Figure 11: Annotated VH and VL sequence of JOVI-1. Hypervariable regions are underlined and highlighted in bold.

Figure 12: Demonstration that peripheral T-cell lymphoma is TRBC-restricted while normal circulating T cells are not. T cells were obtained from the peripheral blood of a patient with circulating T-cell lymphoma cells. Peripheral mononuclear cells were isolated and stained with a panel of antibodies including CD5, TCR, and JOVI-1. Normal and malignant T cells could be distinguished in the cytometer by the intensity of CD5 expression. The bright CD5 (normal T cells) had approximately equal populations of TRBC1 and 2. The intermediate and weak CD5 (tumor) populations were all TRBC2 positive. If this patient had received TRBC2-directed therapy, the lymphoma would have been eradicated and approximately half of the T cells would have been spared.

Figure 13: Demonstration that the JOVI-1-derived VH and VL are correct and that they can fold as a single-chain variable fragment. Parental hybridoma supernatant, recombinant JOVI-1 antibody, and scFv-Fc produced by transfected 293T cells were used to stain a range of cell lines: TCR knockout Jurkat, wild-type Jurkat, TCR knockout Jurkat transduced with TRBC1 TCR in an eBFP2 co-expressing vector, and TCR knockout Jurkat transduced with TRBD2 TCR in an eBFP2 co-expressing vector. Staining was analyzed by flow cytometry. Both recombinant antibody and scFv bound to TRBC2-expressing cells.

Figure 14: JOVI-1-based CARs in various formats. CARs typically contain a binding domain, a spacer, a transmembrane domain, and an intracellular signaling domain. In this study, CARs were generated that contain a JOVI-1 scFv, a spacer derived from either the CD8 stalk, the hinge-CH2-CH3 domain of human IgG1 with mutations that abolish FcR binding; or a spacer derived from human IgG1.

Figure 15: JOVI-1-based CAR function. T cells from healthy donor peripheral blood were transduced with the various CARs described above. T cells were also transduced with a CD19-specific CAR as a control. These T cells were then combined with target cells: TCR knockout Jurkat and wild-type Jurkat and Raji cells (a CD19+ B-cell lymphoma line). Chromium release data are shown for effectors against the various targets. T cells with the JOVI-1 CAR lysed Jurkat cells but not Raji or TCR knockout Jurkat cells.

Figure 16: Self-clearing of T cell cultures with the JOVI-1 CAR. Since T cells contain approximately equal numbers of either TRBC1 or TRBC2 positive T cells, it is possible that some amount of “fratricide” or self-clearing of the culture may occur following CAR administration. This has been shown to occur. In this example, CAR T cells were stained post-transduction and analyzed by flow cytometry. When comparing surrogate-transduced and transduced cells, it can be observed that the T cell population loses TRBC1-positive T cells.

Figure 17: Clonality study of large granular lymphocyte T-cell leukemia (LGL-TL) - Patient A

Figure 18: Clonality study of T-cell large granular lymphocyte leukemia (TL-LGL) - Patient B

Figure 19: Clonality study of large granular lymphocyte T-cell leukemia (LGL-TL) - Patient C

Figure 20: Clonality study of polyclonal T-cell lymphoma (PTCL) - Patient D

Figure 21: Strategies for phage selection based on TRBC peptides . A) Two rounds of solid-phase phage display selection based on TRBC peptides immobilized directly or indirectly on the surface. B) Three rounds of liquid-phase phage display selection based on biotinylated TRBC peptides.

Figure 22: Analysis of polyclonal phage results from phage display selections on TRBC peptides . TRF binding assays using polyclonal phage from solid-phase selections were performed on TRBC peptides directly immobilized as BSA/OA conjugates (A), solid-phase selections on TRBC peptides immobilized on streptavidin/neutravidin (B) and from liquid-phase selections (C).

Figure 23: Schematic representation of the pSANG10-3F vector. The gene encoding the single-chain antibody (scFv) was cloned into the NcoI/NotI site after the T7 promoter and the pelB leader sequence (for periplasmic translocation). The vector also contains a C-terminal hexa-histidine tag (His6) for purification and a tri-FLAG tag for detection.

Figure 24: Primary screening of TRBC1 and TRBC2 specific binding agents . Binding of ninety-four scFvs from TRBC1 (A) and TRBC2 (B) selections to biotinylated TRBC1 and TRBC2 (0.5 μg/ml) immobilized on 96-well Nunc Maxisorp™ plates coated with neutravidin (10 μg/ml). Binding of scFvs to immobilized peptides was detected using europium-conjugated anti-FLAG antibody.

Figure 25: Binding of polyclonal antibody serum from TRBC1-immunized rabbit #13174 to TRBC1 and TRBC2 peptides . (A) After the third immunization (B) After the third immunization and purification of TRBC1-specific antibodies.

Figure 26: Binding of polyclonal antibody serum from rabbit #17363 immunized with TRBC2 peptide to TRBC1 and TRBC2 peptides . (A) After the third immunization (B) After the third immunization and purification of TRBC2-specific antibodies.

Figure 27: Binding of polyclonal antibody serum from rabbit #17364 immunized with TRBC2 peptide to TRBC1 and TRBC2 peptides. (A) After the third immunization (B) After the third immunization and purification of TRBC2-specific antibodies.

ESSENCE OF ASPECTS OF THE INVENTION

The present inventors have developed a method by which it is possible to deplete the population of malignant T cells in an individual without affecting a significant proportion of healthy T cells. In particular, they have developed chimeric antigen receptors (CARs) specific for TRBC1 and TRBC2 for use in the treatment of T cell malignancies.

Thus, in a first aspect, the present invention relates to a chimeric antigen receptor (CAR) that comprises an antigen-binding domain that selectively binds to the constant region 1 of TCR beta (TRBC1) or TRBC2.

In a first embodiment of the first aspect of the invention, a CAR is provided that selectively binds to TRBC1. In this embodiment, the CAR may comprise an antigen-binding domain that has a heavy chain variable region (VH) and a light chain variable region (VL) that comprise the following complementarity determining regions (CDRs):

VH CDR1: SEQ ID No. 7;

VH CDR2: SEQ ID No. 8;

VH CDR3: SEQ ID No. 9;

VL CDR1: SEQ ID No. 10;

VL CDR2: SEQ ID No. 11; and

VL CDR3: SEQ ID No. 12.

The CAR may comprise an antigen-binding domain that has a heavy chain variable region (VH) with the sequence shown as SEQ ID No. 1 and a light chain variable region (VL) with the sequence shown as SEQ ID No. 2.

The CAR may comprise an antigen-binding domain that comprises an scFv with the amino acid sequence shown as SEQ ID No. 3.

The CAR may comprise an amino acid sequence selected from SEQ ID No. 33, 34 and 35.

A CAR may contain CDR3 VH and/or CDR3 VL from those listed in Table 1.

A CAR may comprise an antibody or a functional fragment thereof that contains:

(i) CDR3 of the heavy chain and/or CDR3 of the light chain;

(ii) CDR1, CDR2 and CDR3 of the heavy chain and/or CDR1, CDR2 and CDR3 of the light chain; or

(iii) a heavy chain variable region (VH) and/or a light chain variable region (VL);

from one of the scFvs shown as SEQ ID No. 13 to 22.

In a second embodiment of the first aspect of the invention, a CAR is provided that selectively binds to TRBC2.

In connection with this embodiment, the CAR may comprise VH CDR3 and/or VL CDR3 from those listed in Table 2.

A CAR may comprise an antibody or a functional fragment thereof that contains:

(i) CDR3 of the heavy chain and/or CDR3 of the light chain;

(ii) CDR1, CDR2 and CDR3 of the heavy chain and/or CDR1, CDR2 and CDR3 of the light chain; or

(iii) a heavy chain variable region (VH) and/or a light chain variable region (VL);

from one of the scFvs shown as SEQ ID No. 23 to 32.

In a second aspect, the present invention relates to a nucleic acid sequence encoding a CAR according to the first aspect of the invention.

In a third aspect, a vector is provided which comprises a nucleic acid sequence according to the second aspect of the invention.

In a fourth aspect, a cell is provided that comprises a CAR according to the first aspect of the invention. The cell may be a cytolytic immune cell, such as a T cell or a natural killer (NK) cell.

In a fifth aspect, a method is provided for producing a cell according to the fourth aspect of the invention, which comprises the step of transducing or transfecting the cell with a nucleic acid sequence according to the second aspect of the invention or a vector according to the third aspect of the invention.

In a sixth aspect, a cell according to the fourth aspect of the invention is provided for use in a method of treating T-cell lymphoma or leukemia in an individual, which comprises the step of administering a cell comprising a TCRB1- or TCRB2-selective CAR to the individual in order to cause selective depletion of a population of malignant T cells, together with normal T cells expressing the same TRBC as the malignant T cells, but not to cause depletion of a population of normal T cells expressing TRBC that is not expressed by the malignant T cells.

The method may also include the step of examining the TCR beta constant region (TCRB) of a malignant T cell from the individual to determine whether it expresses TRBC1 or TRBC2.

Also provided is a method of treating T-cell lymphoma or leukemia in an individual, which comprises the step of administering a TCRB1- or TCRB2-selective agent to the individual, wherein the agent causes selective depletion of a population of malignant T cells, along with normal T cells expressing the same TRBC as the malignant T cells, but does not cause depletion of a population of normal T cells expressing TRBC that is not expressed by the malignant T cells.

In a first embodiment of this aspect of the invention, the agent is a TCRB1-selective agent. In a second embodiment of this aspect of the invention, the agent is a TCRB2-selective agent.

The method may also include, prior to the step of administering, the step of examining the TCR beta constant region (TCRB) of a malignant T cell from the individual to determine whether it expresses TRBC1 or TRBC2.

The agent may be a depleting monoclonal antibody or a fragment thereof. The agent may be a conjugated antibody that may contain a chemotherapeutic moiety.

The agent may be a bispecific T cell activator. The agent may be a chimeric antigen receptor (CAR) that is expressed by T cells. The CAR may comprise an amino acid sequence selected from the group consisting of SEQ ID No. 33, 34 and 35.

The agent may comprise the JOVI-1 antibody or a functional fragment thereof.

The agent may comprise an antibody or a functional fragment thereof with a heavy chain variable region (VH) and a light chain variable region (VL) that contain the following complementarity determining regions (CDRs):

VH CDR1: SEQ ID No. 7;

VH CDR2: SEQ ID No. 8;

VH CDR3: SEQ ID No. 9;

VL CDR1: SEQ ID No. 10;

VL CDR2: SEQ ID No. 11; and

VL CDR3: SEQ ID No. 12.

The agent may comprise an antibody or a functional fragment thereof with a heavy chain variable region (VH) with the amino acid sequence shown as SEQ ID No. 1 and a light chain variable region (VL) with the amino acid sequence shown as SEQ ID No. 2.

The agent may comprise a ScFv with the amino acid sequence shown as SEQ ID No. 3. The agent may be provided as a pharmaceutical composition.

T-cell lymphoma or leukemia can be selected from peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS); angioimmunoblastic T-cell lymphoma (AITCL), anaplastic large cell lymphoma (ALCL), enteropathy-associated T-cell lymphoma (EATL), hepatosplenic T-cell lymphoma (HTSL), extranodal NK/T-cell lymphoma-nasal type, cutaneous T-cell lymphoma, primary cutaneous ALCL, T-cell prolymphocytic leukemia, and T-cell acute lymphoblastic leukemia.

The present invention also relates to an agent for use in the treatment of T-cell lymphoma or leukemia according to such a method.

The present invention also relates to a kit containing an agent for use in the above-described capacity.

The present invention also relates to the use of the agent for the production of a medicament for the treatment of T-cell lymphoma or leukemia according to the above-described method.

The present invention also relates to a method for diagnosing T-cell lymphoma or leukemia in an individual, which comprises the step of determining the percentage of T cells that are TRBC1- or TRBC2-positive, out of the total number of T cells in a sample.

A percentage of TRBC1- or TRBC2-positive T cells that is greater than approximately 80% may indicate the presence of T-cell lymphoma or leukemia.

The sample may be a peripheral blood sample or a biopsy.

An agent that binds to all T cells can bind to CD3.

DETAILED DESCRIPTION

The present invention relates to agents, such as chimeric antigen receptors (CARs), that selectively bind to TRBC1 or TRBC2. Such agents are useful in methods of treating T-cell lymphoma or leukemia in an individual. T-cell malignancies are clonal, so they express either TRBC1 or TRBC2. When a TCRB1- or TCRB2-selective agent is administered to an individual, the agent causes selective depletion of a population of malignant T cells, along with normal T cells expressing the same TRBC as the malignant T cells, but does not cause depletion of a population of normal T cells expressing TRBC that is not expressed by the malignant T cells.

TCR β CONSTANT REGION (TRBC)

The T cell receptor (TCR) is expressed on the surface of T lymphocytes and is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. When the TCR interacts with an antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through a series of biochemical events that are mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

The TCR is a membrane-anchored, disulfide-linked heterodimer that is normally composed of highly variable alpha (α) and beta (β) chains expressed as part of a complex with invariant CD3 chain molecules. T cells expressing this receptor are referred to as α:β (or αβ) T cells (~95% of total T cells). A smaller proportion of T cells express an alternative receptor formed by the variable gamma (γ) and delta (δ) chains and are referred to as γδ T cells (~5% of total T cells).

Each α and β chain consists of two extracellular domains: a variable region (V) and a constant region (C), both members of the immunoglobulin superfamily that form antiparallel β-sheets. The constant region is located proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region associates with the peptide/MHC complex (see Figure 1). The constant region of TCR consists of short junctional sequences in which a cysteine residue forms disulfide bonds that connect the two chains.

The variable domains of both the α-chain and β-chain of the TCR have three hypervariable or complementarity-determining regions (CDRs). The variable region of the β-chain also has an additional hypervariable region (HV4), however, it does not normally contact antigen and is thus not considered a CDR.

The TCR also contains up to five invariant γ,δ,ε (collectively referred to as CD3) and ζ chains. The CD3 and ζ subunits mediate TCR signaling through specific cytoplasmic domains that interact with second messenger and adaptor molecules following antigen recognition via αβ or γδ. Expression of the TCR complex on the cell surface is preceded by paired subunit assembly involving both the transmembrane and extracellular domains of the α and β TCR and the γ and δ CD3.

The TCR thus typically consists of a CD3 complex and the TCR α and β chains, which in turn consist of variable and constant regions (Figure 1).

The locus (Chr7:q34) that encodes the TCR β constant region (TRBC) has been duplicated during evolutionary history to yield two virtually identical and functionally equivalent genes: TRBC1 and TRBC2 (Figure 2), which differ in only four amino acids in the mature protein produced by both genes (Figure 3). Each TCR will contain, mutually exclusive, either TRBC1 or TRBC2, and essentially every αβ T cell will express either TRBC1 or TRBC2, mutually exclusive.

The inventors of the present invention have found that, despite the similarity between the sequence of TRBC1 and TRBC2, they can be distinguished. The inventors have also found that the amino acid sequences of TRBC1 and TRBC2 can be distinguished when they are in situ on the surface of a cell, such as a T cell.

MALIGNANT CELLS

The term "malignant" is used herein in its standard meaning to refer to a cell that lacks self-limitation in growth, may be capable of growing into adjacent tissues, and may be capable of spreading to distant tissue. As such, the term "malignant T cell" is used herein to refer to a clonally expanding T cell in relation to lymphoma or leukemia.

The method of the present invention includes determining the TRBC of a malignant T cell. This can be carried out using methods known in the art. For example, it can be determined using PCR, Western blotting, flow cytometry or fluorescence microscopy methods.

Once the TRBC expressed by the malignant T cell is determined, the individual is administered a corresponding TRBC1- or TRBC2-selective agent. "Relevant TRBC-selective agent" means that if the malignant T cell is determined to express TRBC1, then a TRBC1-selective agent is administered, whereas if the malignant T cell is determined to express TRBC2, then a TRBC2-selective agent is administered.

SELECTIVE AGENT

The selective agent binds to either TRBC1 or TRBC2, in a mutually exclusive manner.

As noted above, each αβ T cell expresses a TCR that contains either TRBC1 or TRBC2. In a clonal T cell disorder such as T cell lymphoma or leukemia, malignant T cells derived from a single clone will all express either TRBC1 or TRBC2.

Thus, the present method includes the step of administering a TRBC1- or TRBC2-selective agent to an individual, wherein the agent causes selective depletion of a population of malignant T cells, along with normal T cells that express the same TRBC as the malignant T cells, but does not cause significant depletion of a population of normal T cells expressing a TRBC different from the TRBC of the malignant T cells.

Because a TRBC-selective agent does not significantly deplete the normal T cell population expressing a TRBC different from the malignant T cell, it does not deplete the entire T cell compartment. Sparing a portion of an individual's T cell compartment (i.e., T cells that do not express the same TRBC as the malignant T cells) results in reduced toxicity and decreased cellular and humoral immunodeficiency, and hence decreased risk of infection.

Administration of a TRBC1-selective agent according to the method of the present invention may result in a 5, 10, 20, 50, 75, 90, 95 or 99% depletion, i.e., reduction in the number of T cells expressing TRBC1.

Administration of a TRBC2-selective agent according to the method of the present invention may result in a 5, 10, 20, 50, 75, 90, 95 or 99% depletion, i.e., reduction in the number of T cells expressing TRBC2.

The TRBC1-selective agent may bind to TRBC1 with an affinity that is at least 2-fold, 4-fold, 5-fold, 7-fold, or 10-fold greater than TRBC2. Similarly, the TRBC2-selective agent may bind to TRBC2 with an affinity that is at least 2-fold, 4-fold, 5-fold, 7-fold, or 10-fold greater than TRBC1.

The TRBC1-selective agent causes depletion of a greater proportion of TRBC1-expressing T cells in a cell population than of TRBC2-expressing cells. For example, the ratio of depletion of TRBC1-expressing T cells to TRBC2-expressing cells can be at least 60%:40%, 70%:30%, 80%:20%, 90%:10%, or 95%:5%. Similarly, the TRBC2-selective agent causes depletion of a greater proportion of TRBC2-expressing T cells in a cell population than of TRBC1-expressing cells. For example, the ratio of depletion of TRBC2-expressing T cells to TRBC1-expressing cells can be at least 60%:40%, 70%:30%, 80%:20%, 90%:10%, or 95%:5%.

The method of the invention removes malignant T cells from an individual without affecting a significant proportion of healthy T cells. By "significant proportion" is meant that a sufficient proportion of T cells expressing a TRBC different from the TRBC of the malignant T cells survive to support T cell function in the individual. The agent may cause depletion of less than 20%, 15%, 10%, or 5% of the T cell population expressing a different TRBC.

A selective agent can be selective for either TRBC1 or TRBC2 because it distinguishes between residues as listed below:

(i) N from K at position 3 of TRBC;

(ii) K from N at position 3 of TRBC;

(iii) K from N at position 4 of TRBC;

(iv) N from K at position 4 of TRBC;

(v) F from Y at position 36 TRBC;

(vi) Y from F at position 36 TRBC;

(vii) V of E at position 135 of TRBC; and/or

(viii) E from V at position 135 TRBC.

A selective agent can discriminate between any combination of the above differences.

ANTIBODY

The agent used in the method of the present invention may be a depleting monoclonal antibody (MAB) or a functional fragment thereof, or an antibody mimetic.

The term "depleting antibody" is used in its general sense to refer to an antibody that binds to an antigen present on a target T cell and mediates the death of the target T cell. Administration of a depleting antibody to an individual thus results in a reduction/depletion of the number of cells in the individual expressing the target antigen.

As used herein, "antibody" means a polypeptide with an antigen-binding site that contains at least one complementarity determining region (CDR). An antibody may contain 3 CDRs and have an antigen-binding site that is equivalent to that of a domain antibody (DAT). An antibody may contain 6 CDRs and have an antigen-binding site that is equivalent to that of a classical antibody molecule. The remainder of the polypeptide may be any sequence that provides a suitable framework for the antigen-binding site and positions it appropriately for binding to the antigen. The antibody may be a whole immunoglobulin molecule or a portion thereof, such as a Fab, F(ab)'2, Fv, single chain Fv (ScFv) fragment, or a nanobody. The antibody may be a bifunctional antibody. The antibody may be a non-human, chimeric, humanized, or fully human antibody.

An antibody may thus be any functional fragment that retains the antigen specificity of a full-length antibody.

TRBC1-SELECTIVE ANTIBODIES

An agent for use in the method of the present invention may comprise an antibody or a functional fragment thereof with a heavy chain variable region (VH) and a light chain variable region (VL) that includes one or more of the following complementarity determining regions (CDRs):

VH CDR1: GYTFTGY (SEQ ID No. 7);

VH CDR2: NPYNDD (SEQ ID No. 8);

VH CDR3: GAGYNFDGAYRFFDF (SEQ ID No. 9);

VL CDR1: RSSQRLVHSNGNTYLH (SEQ ID No. 10);

VL CDR2: RVSNRFP (SEQ ID No. 11); And

VL CDR3: SQSTHVPYT (SEQ ID No. 12).

One or more CDRs may or may not each independently contain one or more amino acid mutations (e.g., substitutions) compared to the sequences set forth in SEQ ID Nos. 7 to 12, so long as the resulting antibody retains the ability to selectively bind to TRBC1.

Studies have shown that CDR L3 and H3 are predominantly responsible for high-energy interactions with antigen, thus an antibody or functional fragment thereof may contain CDR3 VH and/or CDR3 VL described above.

Using phage display, several additional antibody binding domains were identified that are highly selective for binding TRBC1 over TRBC2, as described in Example 12.

The agent may comprise an antibody or a functional fragment thereof with a heavy chain variable region (VH) and/or a light chain variable region (VL) that includes one or more complementarity determining regions (CDR3) shown in Table 1.

Table 1

If the agent is a domain antibody, it may contain 3 CDRs, i.e. either CDR1-CDR3 VH or CDR1-CDR3 VL.

The agent may comprise an antibody or a functional fragment thereof that comprises a heavy chain variable region (VH) with the amino acid sequence shown as SEQ ID No. 1 and a light chain variable region (VL) with the amino acid sequence shown as SEQ ID No. 2.

SEQ_ID_1 Jovi-1 VH

SEQ_ID_2 Jovi-1 VL

The agent may comprise an ScFv with the amino acid sequence shown as SEQ ID No. 3.

SEQ_ID_3 Jovi-1 scFv

Alternatively, the agent may comprise an antibody or a functional fragment thereof that includes:

(i) CDR3 of the heavy chain and/or CDR3 of the light chain;

(ii) CDR1, CDR2 and CDR3 of the heavy chain and/or CDR1, CDR2 and CDR3 of the light chain; or

(iii) a heavy chain variable region (VH) and/or a light chain variable region (VL);

from one of the scFvs shown as SEQ ID No. 13-22.

In the sequences shown as SEQ ID Nos. 13-22, the VH and VL portions of the sequence are shown in bold, and the CDR1 and CDR2 sequences for the heavy and light chains are underlined. The CDR3 sequences for VH and VL are shown in Table 1.

SEQ ID No. 13_(CP_01_E09)

SEQ ID No. 14 (CP_01_D12)

SEQ ID No. 15 (CP_01_D10)

SEQ ID No. 16 (CP_01_C08)

SEQ ID No. 17 (CP_01_C11)

SEQ ID No. 18 (CP_01_F03)

SEQ ID No. 19 (CP_01_E07)

SEQ ID No. 20 (CP_01_D03)

SEQ ID No. 21 (CP_01_F06)

SEQ ID No. 22 (CP_01_F02)

TRBC2-SPECIFIC AGENTS

Using phage display, several additional antibody binding domains were identified that are highly selective for binding TRBC2 over TRBC1, as described in Example 12.

The agent may comprise an antibody or a functional fragment thereof with a heavy chain variable region (VH) and/or a light chain variable region (VL) that includes one or more complementarity determining regions (CDR3) shown in Table 2.

Table 2

The agent may comprise an antibody or a functional fragment thereof that includes:

(i) CDR3 of the heavy chain and/or CDR3 of the light chain;

(ii) CDR1, CDR2 and CDR3 of the heavy chain and/or CDR1, CDR2 and CDR3 of the light chain; or

(iii) a heavy chain variable region (VH) and/or a light chain variable region (VL);

from one of the scFvs shown as SEQ ID No. 23-32.

In the sequences shown as SEQ ID Nos. 23-32, the VH and VL portions of the sequence are shown in bold, and the CDR1 and CDR2 sequences for the heavy and light chains are underlined. The CDR3 sequences for VH and VL are shown in Table 2.

SEQ ID No. 23 (CP_03_E05)

SEQ ID No. 24 (CP_03_D05)

SEQ ID No. 25 (CP_03_H06)

SEQ ID No. 26 (CP_03_C12)

SEQ ID No. 27 (CP_03_G02)

SEQ ID No. 28 (CP_03_D04)

SEQ ID No. 29 (CP_03_F10)

SEQ ID No. 30 (CP_03_G09)

SEQ ID No. 31 (CP_03_F09)

SEQ ID No. 32 (CP_03_D09)

Variants of the above amino acid sequences can also be used in the present invention, provided that the resulting antibody binds to TRBC1 or TRBC2 without significant cross-reaction. Typically, such variants have a high degree of sequence identity with one of the above sequences.

Methods for aligning sequences for comparison are well known in the art.

The NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biological Information (NCBI, Bethesda, Md.) and the Internet, for use in conjunction with the blastp, blastn, blastx, tblastn, and tblastx sequence analysis programs. A description of sequence identity determination using this program is available on the NCBI Internet website.

VL or VH domain variants or scFvs typically have at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences set forth in SEQ ID Nos. 1-3, 13-32.

In general, variants may comprise one or more conservative amino acid substitutions compared to a reference amino acid or nucleic acid sequence. Conservative substitutions are those substitutions that do not substantially affect or reduce the affinity of the antibody for binding to TRBC1 or TRBC2. For example, a human antibody that specifically binds to TRBC1 or TRBC2 may comprise up to 1, up to 2, up to 5, up to 10, or up to 15 conservative substitutions in either or both the VH or VL compared to any of the sequences set forth in SEQ ID Nos. 1-3 or 13-32 and retains specific binding to TRBC1 or TRBC2.

Functionally similar amino acids that can be replaced by conservative substitution are well known to those skilled in the art. The following six groups are examples of amino acids that are considered conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

OBTAINING ANTIBODIES

Antibodies can be produced using standard laboratory methods. Antibodies can be obtained from animal serum or, in the case of monoclonal antibodies or their fragments, produced in cell culture. Recombinant DNA technology can be used to produce antibodies according to a well-known method in bacterial or mammalian cell culture.

Methods for producing monoclonal antibodies are well known in the art. Briefly, monoclonal antibodies are typically produced by fusing myeloma cells with spleen cells from a mouse or rabbit that has been immunized with a desired antigen. Herein, the desired antigen is a TRBC1 or TRBC2 peptide, or a TCRβ chain containing either TRBC1 or TRBC2.

Alternatively, antibodies and related molecules, in particular scFv, can be produced outside the immune system by combining libraries of VH and VL chains in a recombinant manner. Such libraries can be constructed and selected using phage display technology, as described in Example 12.

IDENTIFICATION OF TRBC1/TRBC2-SELECTIVE ANTIBODIES

Antibodies that are selective for either TRBC1 or TRBC2 can be identified using methods that are standard in the art. Methods for determining the binding specificity of an antibody include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), Western blotting, immunohistochemistry, flow cytometry, fluorescence resonance energy transfer (FRET) assay, phage display libraries, yeast two-hybrid screens, co-immunoprecipitation, fluorescence bimolecular complementation, and tandem affinity purification.

To identify an antibody that is selective for either TRBC1 or TRBC2, the binding of the antibody to each of TRBC1 and TRBC2 is assessed. Typically, the assessment is made by determining the binding of the antibody to each TRBC separately. An antibody that is selective binds to either TRBC1 or TRBC2, with no significant binding to the other TRBC.

ANTIBODY MIMETICS

The agent may alternatively be a molecule that is not derived from or based on immunoglobulin. A number of "antibody mimetic" designed repeat proteins (DRPs) have been developed to study the binding properties of non-antibody polypeptides.

Repeat proteins such as ankyrin or leucine-rich repeat proteins are ubiquitous binding molecules that, unlike antibodies, occur intra- and extracellularly. Their unique modular architecture features repeating structural units (repeats) that are stacked together to form extended repeat domains, exposing variable and modular target binding surfaces. Based on this modularity, combinatorial libraries of polypeptides with highly diverse binding specificities can be generated. DARPins (engineered ankyrin repeat proteins) are one example of an antibody mimetic based on this technology.

For anticalins, the binding specificity is derived from lipocalins, a family of proteins that perform a variety of functions in vivo related to the physiological transport and storage of chemically sensitive or insoluble compounds. Lipocalins have a robust internal structure containing a highly conserved β-barrel that supports four loops at one end of the protein. These loops for entry into the binding pocket and conformational differences in this part of the molecule are responsible for the variations in binding specificity between different lipocalins.

Avimers are derived from a large family of human extracellular receptor domains using in vitro exon shuffling and phage display, creating multidomain proteins with binding and inhibitory properties.

Versatels are small proteins of 3-5 kDa with >15% cysteines that form a high-density disulfide scaffold, replacing the hydrophobic core present in most proteins. Substitution of a large number of hydrophobic amino acids that make up the hydrophobic core with a small number of disulfides results in a protein that is smaller, more hydrophilic, more resistant to proteases and heat, and has a low density of T-cell epitopes. All four of these properties result in a protein with significantly reduced immunogenicity. Versatels can also be produced in E. coli and are highly soluble and stable.

CONJUGATES

The antibody or mimetic may be a conjugate of the antibody or mimetic and another agent or antibody, for example the conjugate may be a detectable moiety or a chemotherapeutic moiety.

The detectable portion may be a fluorescent moiety, such as a fluorescent peptide. "Fluorescent peptide" refers to a polypeptide that emits light at a detectable wavelength when excited. Examples of fluorescent proteins include, but are not limited to, fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), green fluorescent protein (GFP), enhanced GFP, red fluorescent protein (RFP), blue fluorescent protein (BFP), and mCherry.

A TRBC1- or TRBC2-selective agent conjugated to a detectable moiety can be used to detect TRBC of a malignant T cell.

A chemotherapeutic moiety, as used herein, refers to a moiety that is destructive to a cell and is a moiety that reduces the viability of a cell. A chemotherapeutic moiety may be a cytotoxic drug. Chemotherapeutic agents, considered as non-limiting examples, include alkylating agents, nitrosoureas, ethyleneimines/methylmelamine, alkyl sulfonates, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes such as L-asparaginase; biological response modifiers such as IFNα, IL-2, G-CSF, and GM-CSF; platinum coordination complexes such as cisplatin and carboplatin, anthracenediones, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenal suppressants such as mitotane (o,p'-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol equivalents and ethinyl estradiol; antiestrogen such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone/equivalents; antiandrogens, such as flutamide, gonadotropin-releasing hormone analogues and leuprolide; and nonsteroidal antiandrogens, such as flutamide.

A TRBC-selective agent conjugated to a chemotherapeutic moiety is capable of targeted delivery of the chemotherapeutic moiety to cells that express either TRBC1 or TRBC2.

BISPECIFIC T-CELL ACTIVATORS

A wide range of molecules have been developed based on the basic concept of having two antibody-like binding domains.

Bispecific T-cell activating molecules are a class of bispecific antibody-like molecules that were primarily developed for use as anticancer drugs. They direct the host immune system, more specifically the cytotoxic activity of T cells, against a target cell, such as a cancer cell. In these molecules, one binding domain binds to the T cell via the CD3 receptor and the other binds to target cells, such as a cancer cell (via the tumor-specific molecule). Because the bispecific molecule binds to both the target cell and the T cell, it brings the target cell into proximity with the T cell so that the T cell can exert its effect, such as a cytotoxic effect, on the cancer cell. Formation of the T cell:bispecific Ab:cancer cell complex induces signaling in the T cell. leading, for example, to the release of cytotoxic mediators. Ideally, the agent only induces the desired signaling in the presence of the target cell, resulting in selective lysis.

Bispecific T cell activating molecules have been developed in a number of different formats, but one of the most common is a fusion of two single chain variable fragments (scFvs) from different antibodies. These molecules are sometimes referred to as BiTEs (Bispecific T cell activators).

The agent used in the method of the present invention may be a bispecific molecule that selectively recognizes TRBC1 or TRBC2 and is capable of activating a T cell. For example, the agent may be BiTE. The agent used in the method may comprise:

(i) the first domain that binds to either TRBC1 or TRBC2; and

(ii) a second domain capable of activating T cells.

The bispecific molecule may contain a signal peptide to facilitate its production. The signal peptide may cause secretion of the bispecific molecule by the host cell so that the bispecific molecule can be recovered from the host cell supernatant.

The signal peptide may be located at the amino terminus of the molecule. The bispecific molecule may have the general formula: signal peptide-first domain-second domain.

A bispecific molecule may contain a spacer sequence to connect the first domain to the second domain and spatially separate the two domains.

The spacer sequence may, for example, comprise an IgG1 hinge or a CD8 stalk. The linker may alternatively comprise an alternative linker sequence that has similar length and/or domain spacer properties as the IgG1 hinge or CD8 stalk.

The bispecific molecule may comprise JOVI-1, or a functional fragment thereof, as defined above.

CHIMERIC ANTIGENE RECEPTOR (CAR)

Chimeric antigen receptors (CARs), also known as chimeric T-cell receptors, artificial T-cell receptors, and chimeric immunoreceptors, are engineered receptors that graft arbitrary specificity onto an immune effector cell. In classical CAR, the specificity of a monoclonal antibody is grafted onto a T cell. Nucleic acids encoding the CAR can be transferred into T cells using, for example, retroviral vectors. In this way, large numbers of tumor-specific T cells can be generated for adoptive cell transfer. Phase I clinical trials of this approach have demonstrated efficacy.

The CAR binding domain for the target antigen is typically fused via a spacer and a transmembrane domain to an endodomain that contains or is associated with an intracellular T cell signaling domain. When the CAR binds to the target antigen, it results in the transmission of an activating signal to the T cell expressing the CAR on its surface.

The agent used in the method of the present invention may be a CAR that selectively recognizes TRBC1 or TRBC2. The agent may be a T cell that expresses a CAR that selectively recognizes TRBC1 or TRBC2.

CAR may also contain a transmembrane domain that spans the membrane. It may contain a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which confers good stability to the receptor.

The endodomain is the portion of the CAR involved in signaling. The endodomain either contains or is associated with the intracellular T cell signaling domain. Upon recognition of antigen, the receptors cluster and a signal is transmitted to the cell. The most widely used component for T cell signaling is that of CD3-zeta, which contains three immunoreceptor tyrosine-based activation motifs (ITAMs). It transmits an activation signal to the T cell upon binding to antigen. CD3-zeta may not provide a fully competent activation signal and additional costimulatory signaling may be necessary. For example, chimeric CD28 and OX40 with CD3-zeta can be used to transmit a proliferative/survival signal, or all three can be used together.

The CAR endodomain may contain the CD28 and OX40 endodomain and the CD3-zeta endodomain.

A CAR may contain a signal peptide so that when the CAR is expressed inside a cell, such as a T cell, the nascent protein is directed to the endoplasmic reticulum and then to the cell surface where it is expressed.

The CAR may contain a spacer sequence to connect the TRBC-binding domain to the transmembrane domain and spatially separate the TRBC-binding domain and the endodomain. The flexible spacer allows the TRBC-binding domain to be oriented in different directions to allow TRBC binding.

The spacer sequence may, for example, comprise an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk, or a combination thereof. The linker may alternatively comprise an alternative linker sequence that has similar length and/or domain spacer properties as the IgG1 Fc region, the IgG1 hinge, or the CD8 stalk.

It was found that CARs containing a spacer based on the IgG1 hinge or CD8 stalk showed the best efficacy against Jurkat cells (Figure 15). The spacer may thus contain the IgG1 hinge or CD8 stalk, or a spacer that has similar length and/or domain spacer properties as the IgG1 hinge or CD8 stalk.

The spacer from human IgG1 can be modified to remove Fc-binding motifs.

The CAR may comprise the JOVI-1 antibody or a functional fragment thereof as defined above.

The CAR may comprise an amino acid sequence selected from the group consisting of SEQ ID No. 33, 34 and 35.

>SEQ_ID_33 JOVI-1 CAR with spacer from CD8 stem

METDTLLLWVLLVWIPGSTGEVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFFDFW GQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKL EIKRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPP GGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

>SEQ_ID_34 JOVI-1 CAR with spacer hinge-CH2-CH3pvaa

METDTLLLWVLLVWIPGSTGEVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGAYRFFDFWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVSLGDQASIS CRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPYTFGGGTKLEIKR SDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRL LHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

>SEQ_ID_35 JOVI-1 CAR with spacer from IgG1 hinge

METDTLLLWVLLVWIPGSTGEVRLQQSGPDLIKPGASVKMSCKASGYTFTGYVMHWVKQRPGQGLEWIGFINPYNDDIQSNERFRGKATLTSDKSSTTAYMELSSLTSEDSAVYYCARGAGYNFDGA YRFFDFWGQGTTLTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVSLGDQASISCRSSQRLVHSNGNTYLHWYLQKPGQSPKLLIYRVSNRFPGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQS THVPYTFGGGTKLEIKRSDPAEPKSPDKTHTCPPCPKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRT PIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

In the above CAR sequences, one or more of the 6 CDRs may independently contain or not contain one or more amino acid mutations (e.g., substitutions) compared to the sequences shown as SEQ ID Nos. 7 to 12, as long as the resulting CAR retains the ability to bind to TRBC1.

Variants of the above amino acid sequences may also be used in the present invention, provided that the resulting CAR binds to TRBC1 or TRBC2 and does not have significant cross-reaction. Typically, such variants have a high degree of sequence identity to one of the sequences set forth in SEQ ID No. 33, 34 or 35.

CAR variants typically have at least about 75%, such as at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to one of the sequences set forth as SEQ ID Nos. 33, 34, and 35.

NUCLEIC ACID

The present invention further relates to a nucleic acid encoding an agent such as a BiTE or CAR of the first aspect of the invention.

The nucleic acid sequence may encode a CAR comprising one of the amino acid sequences shown as SEQ ID Nos. 33, 34 and 35.

As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

It should be understood by the skilled person that multiple different polynucleotides and nucleic acids may encode the same polypeptide due to the degeneracy of the genetic code. It should also be understood that the skilled person, using routine methods, can make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to match the codon usage of any particular host organism in which the polypeptides are intended to be expressed.

The nucleic acids of the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that include synthetic or modified nucleotides. A number of different types of modification of oligonucleotides are known in the art. These modifications include methylphosphonate and thiophosphate backbones, the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the uses described herein, it is understood that the polynucleotides may be modified by any method available in the art. Such modifications may be made in order to improve the in vivo activity or life span of the polynucleotides of interest.

The terms "variant", "homolog" or "derivative" with respect to a nucleotide sequence include any substitution, variation, modification, replacement, deletion or addition of one or more nucleic acids in or to the sequence.

VECTOR

The present invention also relates to a vector or a set of vectors that contain one or more nucleic acid sequences of the invention. Such a vector can be used to introduce the nucleic acid sequence into a host cell such that it expresses the CAR of the first aspect of the invention.

The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

The vector may be capable of transfecting or transducing T cells or NK cells.

CELL

The present invention also relates to a cell, such as an immune cell, comprising a CAR according to the first aspect of the invention.

The cell may comprise a nucleic acid or vector of the present invention.

The cell can be a T cell or a natural killer (NK) cell.

T cells can be T cells or T lymphocytes, which are a type of lymphocyte that plays a central role in cellular immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. There are different types of T cells, summarized below.

Helper T cells (Th cells) assist other white blood cells in immunologic processes, including the maturation of B cells into plasma cells and memory B cells and the activation of cytotoxic T cells and macrophages. Th cells express CD4 on the surface. Th cells are activated when peptide antigens are presented to them by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can be one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete a variety of cytokines to facilitate different types of immune responses.

Cytolytic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to MHC class I-associated antigen, which is present on the surface of all nucleated cells. With the help of IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

Memory T cells are a subtype of antigen-specific T cells that persist long after infection has cleared. They rapidly expand to large numbers of effector T cells upon re-presentation of the antigen they recognize, thus providing the immune system with a "memory" against past infections. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), previously known as suppressor T cells, are key to maintaining immune tolerance. Their primary role is to turn off T cell immunity toward the end of the immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

Two main classes of CD4+ Treg cells have been described: natural Treg cells and adaptive Treg cells.

Natural Tregs (also known as CD4+CD25+FoxP3+ Tregs) arise in the thymus and are associated with interactions between developing T cells and myeloid (CD11c+) and plasma cell (CD123+) dendritic cells that have been activated by thymic stromal lymphopoietin (TSLP). Natural Tregs can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent the development of regulatory T cells, causing the lethal autoimmune disease immune dysregulation syndrome, polyendocrinopathy, and enteropathy (IPEX).

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

The cell may be a natural killer (or NK) cell. NK cells form part of the innate immune system. NK cells provide rapid responses to internal signals from virus-infected cells in an MHC-independent manner.

NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGLs) and constitute the third cell type that differentiates from the common lymphoid progenitor that produces B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils, and thymus, where they then enter the circulation.

The CAR cells of the invention may be any of the above-mentioned cell types.

T cells or NK cells expressing the CAR of the first aspect of the invention may be generated ex vivo either from the patient's own peripheral blood (first batch), or in the environment of a hematopoietic stem cell transplant from a donor's peripheral blood (second batch), or from the peripheral blood of an unrelated donor (third batch).

Alternatively, T cells or NK cells expressing the CAR of the first aspect of the invention may be obtained by ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into T or NK cells. Alternatively, an immortalized T cell line may be used that retains its lytic function and could act as a therapeutic agent.

In all of these embodiments, the CAR cells are produced by introducing DNA or RNA encoding the CAR by one of many methods, including transduction with a viral vector, transfection with DNA or RNA.

A cell with a CAR of the invention may be an ex vivo T or NK cell from an individual. The T or NK cell may be from a peripheral blood mononuclear cell (PBMC) sample. The T or NK cells may be activated and/or expanded prior to being transduced with a nucleic acid encoding a CAR of the first aspect of the invention, for example by treatment with a monoclonal antibody against CD3.

The T or NK cell of the invention can be produced by:

(i) isolation of a sample containing a T or NK cell from an individual or from other sources specified above; and

(ii) transducing or transfecting T or NK cells with a nucleic acid sequence/acids encoding a CAR of the invention.

T or NK cells can then be isolated, for example, selected based on expression of the antigen-binding domain of the antigen-binding polypeptide.

The present invention also relates to a kit that comprises a T or NK cell comprising a CAR according to the first aspect of the invention.

PHARMACEUTICAL COMPOSITION

The present invention also relates to a pharmaceutical composition comprising a plurality of cells expressing a CAR according to the first aspect of the invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and/or compounds. Such a formulation may be, for example, in a form suitable for intravenous infusion.

T-CELL LYMPHOMA AND/OR LEUKEMIA

The present invention relates to agents, cells and methods for treating T-cell lymphoma and/or leukemia.

A method for treating T-cell lymphoma and/or leukemia refers to the therapeutic use of an agent. In the present invention, the agent can be administered to an individual who has a disease, T-cell lymphoma and/or leukemia, in order to reduce, decrease, improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

The method of the present invention can be used to treat any lymphoma and/or leukemia associated with clonal expansion of a cell expressing a T-cell receptor (TCR) containing a β constant region. In essence, the present invention relates to a method for treating a disease that includes malignant T cells expressing a TCR that contains a TRBC.

The method of the present invention can be used to treat T-cell lymphoma, in which the malignant T-cell expresses a TCR that contains TRBC. "Lymphoma" is used herein in accordance with its standard meaning in relation to a malignant tumor that typically develops in the lymph nodes, but can also affect the spleen, bone marrow, blood and other organs. Lymphoma is usually present as a solid tumor of lymphoid cells. The primary symptom associated with lymphoma is lymphadenopathy, although secondary (B) symptoms may include fever, night sweats, weight loss, loss of appetite, fatigue, respiratory failure and skin itching.

The method of the present invention can be used to treat T-cell leukemia, in which the malignant T-cell expresses a TCR that contains TRBC. "Leukemia" is used herein in accordance with its standard meaning in relation to a malignant tumor of the blood or bone marrow.

Below is an illustrative, non-exhaustive list of diseases that can be treated by the method of the present invention.

PERIPHERAL T-CELL LYMPHOMA

Peripheral T-cell lymphomas are relatively rare lymphomas, accounting for less than 10% of all non-Hodgkin lymphomas (NHL). However, they are associated with an aggressive clinical course, and the cause and exact cellular origin of most T-cell lymphomas remain to be determined.

Lymphoma usually first appears as a swelling in the neck, armpit, or groin. Additional swelling may occur where other lymph nodes are located, such as the spleen. Typically, enlarged lymph nodes may invade blood vessel spaces, nerves, or the stomach, causing swelling in the arms and legs, tingling, numbness, or a feeling of fullness, respectively. Symptoms of lymphoma also include nonspecific symptoms such as fever, chills, unexplained weight loss, night sweats, lethargy, and itching.

The WHO classification uses morphologic and immunophenotypic hallmarks in combination with clinical aspects and, in some cases, genetics to define a prognostically and therapeutically meaningful classification for peripheral T-cell lymphomas (Swerdlow et al. ; WHO classification of tumors of haematopoietic and lymphoid tissues. 4th ed.; Lyon: IARC Press; 2008). The anatomical locations of neoplastic T cells correspond in part to their putative normal cellular partners and functions and, as such, T-cell lymphomas are associated with lymph nodes and peripheral blood. This approach allows for a better understanding of some of the manifestations of T-cell lymphomas, including cell distribution, some aspects of morphology and even associated clinical outcomes.

The most common T-cell lymphomas are peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), comprising 25% of the total, followed by angioimmunoblastic T-cell lymphoma (AITL) (18.5%).

PERIPHERAL T-CELL LYMPHOMA, NOT SPECIFIED (PTCL-NS)

PTCL-NU comprises more than 25% of all peripheral T-cell lymphomas and NK/T-cell lymphomas, and is the most common subtype. It is diagnosed by exclusion, as it does not correspond to any of the specific mature T-cell lymphomas listed in the current 2008 WHO list. As such, it is analogous to diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NU).

Most patients are adults with a median age of 60 years and a male to female ratio of 2:1. Most cases are nodular in nature, however, extranodal locations occur in approximately 13% of patients and most commonly involve the skin and gastrointestinal tract.

The cytologic spectrum is very broad, ranging from polymorphologic to monomorphologic. Three morphologic distinct variants have been described, including the lymphoepithelioid (Lennert) variant, the T-zone variant, and the follicular variant. The lymphoepithelioid variant of PTCL includes abundant associated epithelioid histiocytes and is usually CD8 positive. It is associated with a better prognosis. The follicular variant of PTCL-NU appears as a potentially distinct clinicopathologic entity.

Most PTCL-NU have a mature T-cell phenotype and most cases are CD4 positive. Seventy-five percent of cases demonstrate variable loss of at least one pan-T-cell marker (CD3, CD2, CD5, or CD7), with CD7 and CD5 most commonly downregulated. CD30 and rarely CD15 may be expressed, with CD15 being an unfavorable prognostic feature. CD56 expression, although rare, also has a negative prognostic impact. Additional unfavorable pathological prognostic factors include a proliferation rate greater than 25% based on KI-67 expression, and the presence of greater than 70% transformed cells. Immunophenotypic analysis of these lymphomas offers little insight into their biology.

ANGIOIMMUNOBLASTIC T-CELL LYMPHOMA (AITCL)

AITC is a systemic disease characterized by a polymorphic infiltrate involving lymph nodes, prominent external endothelial venules (EEVs), and perivascular dilation of follicular dendritic cell (FDC) networks. AITC is considered a de-novo T-cell lymphoma originating from follicular helper-type (TFH) αβ T cells, which are normally located in germinal centers.

AITC is the second most common entity among peripheral T-cell lymphomas and NK/T-cell lymphomas, accounting for approximately 18.5% of cases. It occurs in the middle- to late-life range, with a mean age of 65 years, and approximately equal prevalence in men and women. Clinically, patients typically have late-stage disease with generalized lymphadenopathy, hepatosplenomegaly, and prominent systemic symptoms. A skin rash with associated pruritus is usually present. Polyclonal hypergammaglobulinemia associated with autoimmune phenomena is often present.

Three distinct morphologic patterns have been described in AITCL. The early lesion of AITCL (Pattern I) typically shows preserved architecture with characteristic hyperplastic follicles. The neoplastic proliferation is localized to the periphery of the follicles. In Pattern II, the nodular architecture is partially obliterated with the preservation of a few regressed follicles. The subcapsular sinuses are preserved and even dilated. The paracortical area contains ramified NEVs and there is a proliferation of FDCs outside the B-cell follicle. The neoplastic cells are small to medium in size with minimal cytologic atypia. They often have clear or pale cytoplasm and may demonstrate well-defined cell membranes. A polymorphic inflammatory background is usually evident.

Although AITC is a T-cell malignancy, there is a characteristic proliferation of B cells and plasma cells, which probably reflects the function of the neoplastic cells as TFH cells. Both EBV-positive and EBV-negative B cells are present. Occasionally, the atypical B cells may resemble Hodgkin/Reed-Sternberg-like cells morphologically and immunophenotypically, sometimes leading to diagnostic confusion with this entity. B-cell proliferation in AITC can be extensive, and some patients develop secondary EBV-positive diffuse large B-cell lymphomas (DLBCL) or, more rarely, EBV-negative B-cell neoplasms, often with plasmacytic differentiation.

Neoplastic CD4-positive T cells in AITC show robust expression of CD10 and CD279 (PD-1) and are positive for CXCL13. CXCL13 results in increased B-cell recruitment to lymph nodes via adhesion to NEVs, B-cell activation, plasmacytic differentiation, and spreading of FDC networks; all factors contribute to the morphologic and clinical features of AITC. Intense PD-1 expression in perifollicular tumor cells is particularly useful in distinguishing Pattern I AITC with reactive follicular and paracortical hyperplasia.

Follicular variant PTCL-NU represents a different form with a TFH phenotype. In contrast to AITCL, it lacks prominent NEVs or extrafollicular proliferation of FDC networks. Neoplastic cells may form intrafollicular aggregates, mimicking B-cell follicular lymphoma, but may also have an intrafollicular growth pattern or include expanded mantle zones. Clinically, follicular variant PTCL-NU differs from AITCL because patients more often have early-stage disease with partial lymph node involvement and may lack the systemic symptoms associated with AITCL.

ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)

ALCL can be classified as anaplastic lymphoma kinase (ALK)+ or ALCL-ALK-.

ALCL-ALK+ is one of the best characterized forms of peripheral T-cell lymphoma, with characteristic "hallmark cells" with horseshoe-shaped nuclei and expressing ALK and CD30. It accounts for approximately 7% of all peripheral T-cell and NK-cell lymphomas and is most common in the first three decades of life. Patients often present with lymphadenopathy, but extranodal involvement (skin, bone, soft tissue, lung, liver) and B-features are common.

ALCL-ALK+ displays a broad morphologic spectrum, with five distinct patterns described, but all variants contain some distinctive cells. Distinctive cells have eccentric horseshoe- or kidney-shaped nuclei, and prominent perinuclear eosinophilic Golgi regions. Tumor cells grow in a linked fashion with a predilection for sinus involvement. Small tumor cells predominate in the small cell variant, and in the lymphohistiocytic variant, the abundance of histiocytes masks the presence of tumor cells, many of which are small.

By definition, all cases are ALK and CD30 positive, typically with weaker expression in small tumor cells. Loss of pan-T-cell markers is common, with loss of CD3 surface expression in 75% of cases.

ALK expression results from a characteristic recurrent genetic alteration consisting of translocation of the ALK gene on chromosome 2p23 to one of many partner genes, resulting in expression of a chimeric protein. The most common partner gene, occurring in 75% of cases, is the nucleophosmin gene ( NPM1 ) on chromosome 5q35, resulting in t(2;5)(p23;q35). The cellular distribution of ALK in different translocation variants may vary depending on the partner gene.

ALCL-ALK- is included as a provisional category in the 2008 WHO classification. It is defined as a CD30-positive T-cell lymphoma that is morphologically indistinguishable from ALCL-ALK+ with a linked growth pattern and the presence of distinctive cells but lacking ALK protein expression.

Patients are typically adults between 40 and 65 years of age, in contrast to ALCL-ALK+, which is more common in children and young adults. ALCL-ALK- may involve both nodal and extranodal tissues, although the latter is less common than in ALCL-ALK+. Most cases of ALCL-ALK- demonstrate a lymph node architecture with folds of cohesive neoplastic cells with typical "hallmark" features. Unlike ALCL-ALK+, a small cell morphologic variant is not recognized.

In contrast to the ALK+ form, ALCL-ALK- shows greater preservation of T-cell marker expression, while expression of cytotoxic markers and epithelial cell surface antigen (EMA) is less likely. Gene expression profiles and recurrent chromosomal imbalances are different in ALCL-ALK- and ALCL-ALK+, confirming that they are distinct forms at the molecular and genetic level.

ALCL-ALK- is clinically distinct from both ALCL-ALK+ and PTCL-NU, with significant differences in prognosis between the three distinct entities. The reported 5-year overall survival rate of ALCL-ALK- is 49%, which is not as good as that of ALCL-ALK+ (around 70%), but is significantly better than that of PTCL-NU (32%).

ENTEROPATHY ASSOCIATED T-CELL LYMPHOMA (TCL)

TLE is an aggressive neoplasm thought to originate from intestinal intraepithelial T cells. The 2008 WHO classification distinguishes two morphologically, immunohistochemically, and genetically distinct types of TLE: Type I (representing the majority of TLE) and Type II (accounting for 10–20% of cases).

Type I TLE is typically associated with overt or asymptomatic gluten-sensitive enteropathy and is more commonly seen in patients of northern European origin due to the high prevalence of celiac disease in this population.

Most commonly, TCLE plaques are found in the jejunum or ileum (90% of cases), with rare cases in the duodenum, colon, stomach, or extragastrointestinal sites. Intestinal lesions are typically multifocal with mucosal ulceration. The clinical course of TCLE is aggressive, and most patients die of the disease or complications of the disease within one year.

The cytologic spectrum of type I TCLE is broad, and some cases may contain anaplastic cells. A polymorphic inflammatory background is present, which in some cases may obscure the neoplastic component. The intestinal mucosa in areas adjacent to the tumor often shows features of celiac disease, with “blunting” of the villi and increased numbers of intraepithelial lymphocytes (IELs), which may represent damaged progenitor cells.

According to immunohistochemistry, neoplastic cells are often CD3+CD4-CD8-CD7+CD5-CD56-βF1+ and contain granule-associated cytotoxic proteins (TIA-1, granzyme B, perforin). CD30 is partially expressed in almost all cases. CD103, which is a mucosal homing receptor, may be expressed in TCLE.

Type II ITC, also referred to as monomorphic CD56+ intestinal T-cell lymphoma, is defined as a tumor of the intestine that is composed of small to medium-sized monomorphic T cells expressing both CD8 and CD56. Lateral intramucosal extension of the tumor is often present, and there is no inflammatory background. Most cases express the γδ TCR, although there are cases associated with the αβ TCR.

Type II T-cell lymphoma has a more widespread worldwide distribution than Type I T-cell lymphoma and is often seen in Asian or Latino populations, where celiac disease is rare. In individuals of European descent, type II T-cell lymphoma accounts for approximately 20% of intestinal T-cell lymphomas, with a history of celiac disease in at least a subset of cases. The clinical course is aggressive.

HEPATOSPLENIC T-CELL LYMPHOMA (HTCL)

HSTL is an aggressive systemic neoplasm primarily derived from γδ cytotoxic T cells of the innate immune system, although in rare cases it may also originate from αβ T cells. It is one of the rarer T-cell lymphomas and typically affects adolescents and young adults (median age, 35 years) with a strong male predominance.

EXTRANODAL NK/T-CELL LYMPHOMA OF THE NASAL TYPE

Extranodal NK/T-cell lymphoma of the nasal type is an aggressive disease, often with destructive midline lesions and necrosis. Most cases are NK cell-derived, but some are cytotoxic T cell-derived. It is universally associated with Epstein-Barr virus (EBV).

T-CELL LYMPHOMA OF THE SKIN

The method of the present invention can also be used to treat cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is characterized by the migration of malignant T cells to the skin, causing a variety of lesions. These lesions change shape as the disease progresses, typically first appearing as a rash and eventually forming plaques and tumors before metastasizing to other parts of the body.

Cutaneous T-cell lymphomas include those mentioned in the following illustrative, non-exhaustive list: mycosis fungoides, reticulosis ascendens, Sezary syndrome, granulomatous laxative, lymphomatous papulosis, pityriasis lichenoides chronicus, CD30+ cutaneous T-cell lymphoma, secondary cutaneous CD30+ large cell lymphoma, CD30- cutaneous large T-cell lymphoma without mycosis fungoides, pleomorphic T-cell lymphoma, Lennert lymphoma, subcutaneous T-cell lymphoma, and lymphangioma.

The signs and symptoms of CTCL vary depending on the specific disease, of which the two most common types are mycosis fungoides and Sezary syndrome. Classic mycosis fungoides is divided into three stages:

Patch (atrophic or non-atrophic): Non-specific dermatitis, patches on the lower trunk and buttocks; minimal/no itching;

Plaque: intensely pruritic plaques, lymphadenopathy; and

Tumor: Tends to ulcerate.

Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include edematous skin, lymphadenopathy, palmar and/or plantar hyperkeratosis, alopecia, nail dystrophy, eyelid eversion, and hepatosplenomegaly.

Of all primary cutaneous lymphomas, 65% are T-cell type. The most common immunophenotype is CD4-positive. There is no common pathophysiology for these diseases, as the term cutaneous T-cell lymphoma covers a broad spectrum of disorders.

The primary etiologic mechanisms for the development of cutaneous T-cell lymphoma (eg, mycosis fungoides) are unclear. Mycosis fungoides may be preceded by chronic T-cell-mediated inflammatory skin disease, which may occasionally progress to fatal lymphoma.

PRIMARY CUTANEOUS ALCL (C-ALCL)

C-ALCL is often indistinguishable from ALC-ALK- in morphology. It is defined as a cutaneous large cell neoplasm with anaplastic, pleomorphic, or immunoblastic morphology with >75% of cells expressing CD30. Together with lymphomatoid papulosis (LP), C-ALCL belongs to a spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders that as a group comprises the second most common group of cutaneous T-cell lymphoproliferations after mycosis fungoides.

The immunohistochemical staining profile is very similar to ALCL-ALK-, with a higher proportion of cases staining positive for cytotoxic markers. At least 75% of tumor cells should be positive for CD30. CD15 may also be expressed, and when lymph node involvement occurs, differential diagnosis with classical Hodgkin lymphoma may be difficult. Rare cases of ALCL-ALK+ may occur with cutaneous lesions, and may resemble C-ALCL.

T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 15% and 25% of ALL in pediatric and adult cohorts, respectively. Patients typically have high white blood cell counts and may exhibit organomegaly, particularly mediastinal enlargement, and CNS involvement.

The method of the present invention can be used to treat T-ALL, which is associated with a malignant T cell expressing a TCR that contains TRBC.

T-CELL PROLYMPHOCYTIC LEUKEMIA

T-cell prolymphocytic leukemia (T-PLL) is a leukemia of mature T cells with aggressive behavior and a predilection for the blood, bone marrow, lymph nodes, liver, spleen, and skin lesions. T-PLL primarily affects adults over age 30. Other names include T-cell chronic lymphocytic leukemia, "nodular type" T-cell leukemia, and T-prolymphocytic leukemia/T-cell lymphocytic leukemia.

In peripheral blood, T-PLL consists of medium-sized lymphocytes with single nucleoli and basophilic cytoplasm with occasional cavities or processes. The nucleoli are typically round to oval, and occasional patients have cells with more irregular nuclear borders that resemble the medulla seen in Sézary syndrome. The small cell variant accounts for 20% of all T-PLL cases, and the Sézary-like (medulla) variant is seen in 5%.

T-PLL has the immunophenotype of a mature (post-thymic) T lymphocyte, and neoplastic cells are typically positive for the pan-T antigens CD2, CD3, and CD7 and negative for TdT and CD1a. The CD4+/CD8- immunophenotype is present in 60% of cases, the CD4+/CD8+ immunophenotype is present in 25%, and the CD4-/CD8+ immunophenotype is present in 15% of cases.

PHARMACEUTICAL COMPOSITION

The method of the present invention may include the step of administering the agent in the form of a pharmaceutical composition.

The agent can be administered with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The pharmaceutical carrier, excipient or diluent can be selected taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions can contain as a carrier, excipient or diluent, or in addition to a carrier, excipient or diluent, any suitable binding agent(s), lubricant(s), suspending agent(s), coating agent(s), solubility enhancing agent(s) and other carriers.

INTRODUCTION

The agent may be administered by any of a number of routes that make the active ingredient bioavailable. For example, the agent may be administered orally, parenterally, intraperitoneally, intravenously, subcutaneously, transdermally, intramuscularly, by local delivery such as through a catheter or stent.

Typically, the physician will determine the actual dosage that will be most appropriate for the individual, and this will vary depending on the age, weight, and response of the individual patient. The dosage is such that it is sufficient to reduce or deplete the number of clonal T cells expressing either TRBC1 or TRBC2.

APPLICATION

The present invention also relates to an agent for use in the treatment of T-cell lymphoma according to the method of the first aspect. The agent may be any agent defined above.

The present invention also relates to the use of an agent as defined above in the manufacture of a medicament for the treatment of T-cell lymphoma according to the method of the first aspect.

KIT

The present invention further relates to a kit comprising the agent defined above for use in the treatment of T-cell lymphoma according to the method of the first aspect.

The kit may also contain reagent(s) suitable for detecting TRBC of a malignant T cell. For example, the kit may contain PCR primers or antibody(ies) that are specific for either TRBC1 or TRBC2.

METHOD FOR DETERMINING T-CELL LYMPHOMA AND/OR LEUKEMIA

The present invention further relates to a method for determining the presence of T-cell lymphoma or leukemia in an individual, comprising the step of determining the proportion of T cells in a sample from the individual that are either TRBC1- or TRBC2-positive.

T-cell lymphomas involve clonal expansion of individual malignant T cells. As such, the presence of T-cell lymphoma in an individual can be detected by determining the proportion of T cells with either TRBC1 or TRBC2 in a sample obtained from the patient.

The sample may be a peripheral blood sample, a lymph sample, or a sample taken directly from the tumor, such as a biopsy sample.

The proportion of T cells out of the total that are TRBC1- or TRBC2-positive, indicating the presence of T-cell lymphoma or leukemia, may be, for example, 80, 85, 90, 95, 98, or 99% of the total cell population.

The method may include determining the infiltration of individual T cell populations in a biopsy or sample. In the present invention, the presence of T cell lymphoma or leukemia is indicated if 80, 85, 90, 95, 98 or 99% of the total T cell population in the sample are either TRBC1 or TRBC2.

The total number of T cells in a sample can be determined by determining the number of cells in the sample expressing CD3, CD4, CD8 and/or CD45. A combination of these markers can also be used.

The proportion of total T cells in a sample that express either TRBC1 or TRBC2 can be determined using methods known in the art, such as flow cytometry, immunohistochemistry, or fluorescence microscopy.

The invention will now be further described by way of examples, which are intended to assist a person skilled in the art in carrying out the invention and in no way limit the scope of the invention.

EXAMPLES

Example 1 - Recognition of TRBC1 and TRBC2-expressing cells

The JOVI-1 antibody was previously described by Viney et al. (Hybridoma; 1992; 11(6); 701-713) and is commercially available (Abcam, ab5465). The present inventors have determined that JOVI-1 is capable of recognizing cells based on specific expression of TRBC1 or TRBC2.

We constructed two plasmid vectors delivering complete TCR variable and constant regions that differ only in the expression of either TRBC1 or TRBC2. These plasmids were used to generate retroviral supernatant by transient transfection of 293T cells. This supernatant was used to stably transduce Jurkat TCR knockout T cells (a T-ALL cell line with a mutation in the TCR beta chain locus that prevents expression of this chain and thus the complete TCR/CD3 surface complex). This resulted in cell lines that are identical except for the expression of either TRBC1 or TRBC2. Staining of these cell lines revealed complete expression of the TCR/CD3 surface complex, and only cells expressing TRBC1 were stained with the JOVI-1 antibody (Figure 4).

Example 2 CD4+ and CD8+ T cells from a healthy donor contain separate TRBC1-positive and TRBC1-negative populations

We tested the JOVI-1 antibody on primary human T cells from healthy donors. These analyses revealed that all donors had proportions of both CD4+ and CD8+ T cells that expressed TRBC1, and proportions of each that did not. Approximately 20-50% of normal CD4+ and CD8+ T cells are TRBC1-positive (Figures 6 and 7).

Example 3 - Clonal T cell lines expressing TCR are TRBC1 positive or negative

Cell lines were derived from the patient's original clonal tumor population. Staining of TCR-expressing T cell lines revealed that T cells expressed either TRBC1 or TRBC2, confirming that it is a marker of clonality. Of the three T cell lines tested, Jurkat cells (known as TRBC1+), but not HPB-ALL or HD-Mar-2 cells (known as TRBC2+), stained with JOVI-1, confirming exclusive expression of either TRBC1 or TRBC 2 (Figure 8).

Example 4 - Primary clonal T cells in patients with T-prolymphocytic leukemia are TRBC1-positive or negative

Clonal T cells isolated from the peripheral blood of patients with T-prolymphocytic leukemia (T-PLL) are either uniformly TRBC1-positive or TRBC1-negative.

Example 5 - Effect of mutation of residues that are unique to TCBC1

TCR-encoding plasmid vectors were generated that were identical except for the TRBC1/2 hybrid mutations in the TCR β-chain constant region. Analysis showed that JOVI-1 recognizes differences in residues at positions 3 and 4 of the TCRβ constant chain, indicating that these residues are accessible to antibody recognition and are likely the best targets for generating agents that discriminate TRBC1 from TRBC2, or TRBC2 from TRBC1 (Figure 5).

Example 6 - Specific lysis of TRBC1- but not TRBC2-TCR-expressing T cells.

Wild-type Jurkat T cells (CD34-, TRBC1+) were mixed with TCRαβ knockout Jurkat T cells transduced with TRBC2 co-expressing the CD34 marker gene (CD34+TRBC2+). These cells were incubated with JOVI-1 alone or incubated with JOVI-1 and complement for 1 hour. Cells were washed and stained for CD34, annexin V and 7-AAD. Cells were analyzed by flow cytometry.

Figure 9 shows CD34 expression in live populations defined as annexin-V negative and 7-AAD weak expressing populations. Selective lysis of TRBC1 (CD34-) T cells was observed (Figure 9).

Wild-type Jurkat T cells are normally TRBC1+ and do not express the truncated CD34 marker gene. As described above, we generated a TRBC2+ Jurkat line by transducing TCRαβ knockout Jurkat T cells with a retroviral vector that encodes the TRBC2 TCR as well as the truncated CD34 marker gene. These T cells were then mixed together. We then incubated the T cells with either JOVI-1 alone or with JOVI-1 and complement for 1 hour. For convenience, we could distinguish TRBC1 and TRBC2 populations by staining for the CD34 marker gene and thus avoided undetectable TRBC1 TCR due to TCR internalization after prolonged incubation with anti-TCR mAb. Cells were washed and stained for CD34, annexin V, and 7-AAD. The cells were analyzed by flow cytometry. By gating live cells (i.e., cells that were negative for annexin V and had low 7-AAD brightness), we were able to determine that TRBC1 T cells were selectively lysed by JOVI-1 in the presence of complement (Figure 9).

Example 7 - Epstein-Barr virus (EBV)-specific polyclonal T cells can be divided into two approximately equal TRBC1/2 populations.

Peripheral blood T cells were obtained from a healthy blood donor. Mononuclear cells were isolated and most cells were cryopreserved. A small number of cells were infected with a laboratory strain of EBV (B95-8). After several weeks, an immortalized EBV-infected cell line, known as a lymphoblastic cell line (LCL), was generated. This cell line is known to represent a large collection of different EBV antigens. Previously cryopreserved mononuclear cells were thawed and restimulated with this LCL line weekly for 4 weeks in the presence of IL2. This method selectively expands EBV-specific T cells from the peripheral blood mononuclear population. This method is known to result in a polyclonal line where >90% of the T cells are EBV-specific and reflect donor immunity to EBV. Specificity was verified by demonstrating high levels of lysis of autologous LAK cells but not allogeneic LAK or K562 cells (Figure 10a). This cell line was then stained with JOVI-1 and shown to contain an approximately equal mixture of TRBC1 and TRBC2 T cells (Figure 10b).

Thus, if a therapeutic agent is administered that depletes either the TRBC-1 or TRBC2 compartment, adequate immunity to EBV will be preserved. Since immunity to EBV is considered a model immune response system, it is reasonable to assume that immunity to other pathogens will be equally preserved.

Example 8 - JOV1 staining of circulating peripheral T-cell lymphoma

The hypothesis was that T-cell lymphomas, being clonal, would express either TRBC1 or TRBC2 T-cell receptors, whereas normal T cells, being polyclonal, would comprise a T-cell population that was a mixture of TRBC1- or TRBC2-positive T cells. To demonstrate this, a T-cell lymphoma blood sample was obtained from a patient whose lymphoma was circulating in the peripheral blood. Peripheral blood mononuclear cells were isolated and stained with a panel of antibodies that included CD5 and JOVI1. The total T-cell population (which contains both lymphoma and normal T cells) was first identified. This population contained T cells with normal (strong) CD5 expression and T cells with intermediate/weak CD5 expression. The former represent normal T cells, whereas the latter represent lymphoma. Binding to JOVI-1 was further examined and the results are shown in Figure 12.

Intermediate and weak CD5 (tumor) populations were all TRBC2 positive.

Example 9 - Detection of JOVI-1 VH/VL sequences

Using rapid amplification of cDNA ends (5' RACE) with primers that anneal to the mouse IgG CH1 constant region and the mouse kappa constant region, we isolated a single functional VH sequence and a single functional VL sequence from the JOVI-1 hybridoma. The VH and VL sequences are SEQ ID NOs. 1 and 2, respectively (see above). The annotated VH and VL sequence is shown in Figure 11.

These VH and VL sequences were cloned back in frame with mouse IgG heavy chain and kappa light chain, respectively. Furthermore, VH and VL were fused to form a single-chain variable fragment (scFv), which was then fused to the hinge-CH2-CH3 region of mouse IgG2a to create scFv-Fv. The amino acid sequence of scFv is provided in the detailed description as SEQ ID No. 3. The recombinant antibody and recombinant scFv-Fc were produced by transfection of 293T cells. Together with JOVI-1 from hybridoma, the following cells were stained: TCR knockout Jurkat cells; wild-type Jurkat cells; TCR knockout Jurkat cells transduced with TRBC1 co-expressing eBFP2 and TCR knockout Jurkat cells transduced with TRBC2 co-expressing eBFP2. Both the recombinant antibody and the JOVI-derived scFv-Fc bound TRBC2, confirming that we had identified the correct VH/VL and that the JOVI-1 VH/VL can be folded as an scFv. These binding data are shown in Figure 13.

Example 10 - CAR function based on JOVI-1

JOVI-1 scFvs were cloned into CAR formats. To determine which spacer length would result in an optimal JOVI-1 CAR, third generation CARs were generated with either a human Fc spacer, a human CD8 stalk spacer, or an IgG1 hinge-derived spacer (Figure 4). Primary human T cells from normal donors were transduced with these CARs and lysis of Jurkat cells and TCR knockout Jurkat cells was compared. JOVI-1 scFv CARs that had either an IgG1 hinge or CD8 stalk spacer lysed Jurkat cells but not TCR knockout Jurkat cells (Figure 5), demonstrating the expected specificity. Since healthy donor CAR-transduced T cells should have a TRBC 1/2 T cell mixture, it was expected that the cultures would “self-clear.” In fact, this is what was observed. JOVI-1 staining of CAR T cell cultures that became 100% TRBC2 negative is shown in Figure 6.

MATERIALS AND METHODS

DEMONSTRATION OF JOVI-1 SPECIFICITY

A tricistronic retroviral cassette encoding the well-characterized human TCR and a suitable marker gene was generated. Coding sequences for the TCRα and β chains were generated by de novo gene synthesis from overlapping oligonucleotides. These chains were linked in frame to the FMDV 2A peptide for co-expression. A truncated CD34 marker gene was cloned from cDNA by PCR and co-expressed with the TCR chains using an internal ribosome entry site (IRES). The cassette was inserted into a retroviral vector. Variants of this construct were generated by linking overlapping PCR with primers that introduced the desired mutations. The accuracy of the constructs was confirmed by Sanger sequencing. Jurkat cell line 76 is a well-characterized derivative of the Jurkat T cell line, knocked out in both TCR chains, α and β. This Jurkat line was transduced with the above retroviral vectors using standard methods.

STAINING AND ANALYSIS OF JURCAT CELLS, PERIPHERAL BLOOD T CELLS AND CELL LINES

Jurkat cells were obtained from the European Collection of Animal Cell Cultures (ECACC) and constructed as described above. Other T cell lines were also obtained from ECACC. Peripheral blood was collected by venipuncture from healthy donors. Blood was treated with Ficoll to isolate mononuclear cells. Cells were stained with JOVI-1 as well as commercially available monoclonal antibodies that recognize all TCRs and CD3. In the case of engineered T cells, cells were stained with antibodies that recognize CD34. In the case of peripheral blood mononuclear cells, cells were stained with antibodies that recognize CD4 and CD8. Antibodies were purchased conjugated to appropriate fluorophores so that independent fluorescent signals could be obtained with simultaneous analysis of cells in a flow cytometer.

DEMONSTRATION OF SPECIFIC T-CELL LYSIS WITH TRBC1

Wild-type Jurkat T cells (TRBC1 - TCR) and TRBC2 TCR engineered Jurkat TCR KO T cells were mixed together in a 1:1 ratio. This mixture of Jurkat cells was then incubated with the monoclonal antibody JOVI-1 at a concentration of 1 μg/ml in the absence or presence of complement. Four hours later, the cells were stained with annexin-V and 7AAD, and CD34. Conveniently, the CD34 marker gene can distinguish between wild-type (TRBC1) and transgenic (TRBC2) Jurkat cells. Cell populations were analyzed by flow cytometry. Live cells were selectively examined by gating cytometric events that were negative for annexin-V and had a weak brightness for 7AAD. In this way, the survival of transgenic T cells (TRBC2) was investigated compared with wild-type (TRBC1) T cells.

Example 11 - Study of clonality of T-cell lymphoproliferative disorders

Four patients, three with T-cell lymphoproliferative disorder with large granular lymphocytes (TL-LGL) and one with peripheral T-cell lymphoma (PTCL), were tested to confirm that the malignant cells were uniformly either TRBC1-positive or negative.

Whole blood or bone marrow was collected from patients with T-lymphoproliferative disorders. Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll gradient centrifugation. Freshly isolated PBMCs were pelleted and stained for 20 min with the appropriate preconjugated antibodies. Cells were then washed and resuspended in phosphate-buffered saline and immediately analyzed by flow cytometry on a BD LSR Fortessa II. Viable lymphocytes were identified by forward/side scatter (FSc/SSc) properties and by failure to uptake dye to detect dead cells. T cells were identified by staining with anti-TCR alpha/beta antibody. Tumor and normal T cell populations were identified using appropriate cell surface dyes for each sample, depending on the immunophenotype previously determined by clinical laboratory analysis.

The results are shown in figures 18 to 21.

In patient A (TL-BGL, Figure 18), normal T cells were CD7 bright, and contained a mixture of CD4/CD8 cells and a mixed population of TRBC1+ or TRBC1- cells. In contrast, malignant cells were CD7- or CD7-weak, were uniformly CD8+CD4-, and were uniformly TRBC1-.

In patient B (TL-BGL, Figure 19), malignant cells were identified as CD4-, CD8+, CD7+ CD57+ and were clonally TRBC1- (highlighted panel). Normal CD4+CD8- and CD4-CD8+ T cells contained TRBC1+ and TRBC1- populations.

In patient C (TL-BGL, Figure 20), normal CD4+ and CD8+ T cell populations were 30-40% TRBC1+. Malignant cells were identified as CD4-, CD8+, CD7+ CD57+ and were clonally TRBC1+ (highlighted panel, note that 84% of cells are TRBC1, the remaining 16% are likely contaminated with “normal” T cells). Normal CD4+CD8- and CD4-CD8+ T cells contained TRBC1+ and TRBC1- populations.

In patient D (PTCL-NU, Figure 21), malignant cells in the bone marrow, as detected by high forward scatter (FSC) and low brightness of CD5 and CD4, were homogeneously TRBC1+, while CD4+CD8- and CD4-CD8+ T cells contained both TRBC1+ and TRBC1- populations.

Example 12 - Production of human monoclonal antibodies that discriminate between two isoforms of the constant domain of the T-cell receptor β-chain using phage display

To generate antibodies that differentiate between TRBC2 and TRBC1, peptide fragments were synthesized covering the region of difference between the two TRBC isoforms. Of the four amino acid differences between TRBC2 and TRBC1, two were found at the beginning of the constant domains. Peptides (see below) representing these regions were synthesized and used to generate antibodies.

TRBC2 VLEDLKNVFPPEVAV (SEQ ID No. 36)

TRBC1 VLEDLNKVFPPEVAV (SEQ ID No. 37)

These peptides were prepared in biotinylated, non-biotinylated and cysteine-modified forms (by adding a cysteine at the C-terminus). The cysteine-modified forms of TRBC1 and TRBC2 were then conjugated to modified bovine serum albumin (Imm-Link BSA, Innova 462-001) or ovalbumin (Imm-Link Ovalbumin, Innova 461-001) according to the conditions recommended by the manufacturer.

Results

Phage display antibody selections

Human phage display libraries were constructed and phage selections were performed as described in (Schofield et al., 2007 Genome Biol8, R254). To identify TRBC1- and TRBC2-specific antibodies in the antibody library, multiple rounds of phage display selections were performed. Two phage selection strategies were used in parallel to maximize the chance of obtaining a large panel of specific binders. These strategies are known as solid-phase selection and liquid-phase selection (Figure 21). In solid-phase selection, phage antibodies are allowed to bind to a target antigen immobilized on a solid surface (Schofield et al., 2007, as above). In liquid-phase selection, phage antibodies bind to biotinylated antigen in solution, and the phage antibody-antigen complex is then captured on paramagnetic beads coated with streptavidin or neutravidin. Within the solid-phase selection strategy, two different approaches of antigen immobilization or presentation were used. In the first approach, TRBC peptides conjugated to bovine serum albumin (BSA) or ovalbumin (OA) were immobilized in Maxisorp™ immunotubes by direct absorption. In the second approach, biotinylated TRBC peptides were indirectly immobilized in Maxisorp™ immunotubes that were pre-coated with streptavidin or neutravidin.

In order to select antibodies that are specific for the desired peptide, all selections were performed in the presence of an excess of the opposite peptide. For example, all TRBC1 selections were performed in the presence of a 10-fold molar excess of non-biotinylated TRBC2. This method is known as "deselection" and was expected to deplete antibodies that recognize common epitopes on both TRBC peptides, since these antibodies preferentially bind to excess TRBC2 in solution. To avoid enrichment of clones with antibodies that bind to the carrier protein (BSA or OA) or the immobilization partner (streptavidin or neutravidin from Thermo Fisher Scientific), a combination of the two strategies was used.

The first strategy involved changing the conjugation or immobilization partner between rounds of selection. For directly immobilized peptides, the first round of selections was performed on a BSA-peptide, and an OA-peptide conjugate was used for round 2. Similarly, biotinylated TRBC peptides were immobilized on streptavidin in round 1, and neutravidin was used for immobilization in round 2.

The second strategy was to deplete the phage library of any binders to the conjugation/immobilization partners by performing "deselection" in round 1. For directly immobilized peptides, "deselection" was performed using a phage-peptide coupling step in the presence of a 10-fold molar excess of free BSA in solution. For biotinylated peptides immobilized on streptavidin, the phage library was pre-incubated with paramagnetic beads coated with streptavidin. The beads were removed before adding the phage to the antigen tubes, thus limiting the streptavidin binder from being selected. The different selection conditions used are summarized in Figure 21. See Table 3 for details of the selection conditions.

Polyclonal phage resulting from round 2 selection were tested by ELISA using different presentations of peptides or accessory proteins. These included TRBC peptides directly immobilized as BSA or OA conjugates or biotinylated peptides indirectly immobilized on streptavidin or neutravidin. Control proteins included streptavidin, neutravidin, BSA and an unrelated antigen. Phage binding was detected with a mouse anti-M13 antibody (GE healthcare) followed by europium-labeled anti-mouse Fc antibody (Perkin Elmers) using time-resolved fluorescence (TRF) (Figure 22). This result demonstrates preferential binding of polyclonal phage populations to the corresponding TRBC peptide (compared to the opposite TRBC peptide). For example, polyclonal phage obtained by selection for TRBC1 showed significantly higher binding signal to TRBC1 than to TRBC2, and vice versa. There was little or no binding to immobilization or conjugation partners and to an unrelated antigen.

Subcloning of single-chain antibody (scFv) and monoclonal screening

The scFv populations from round 2 and round 3 selection were subcloned into the pSANG10-3F expression vector and transformed into E. coli BL21 (DE3) cells. 1128 individual transformants (564 clones/TRBC peptide) were plated in 12×96-well culture plates (94 clones/plate) and antibody expression was induced using autoinduction medium. Recombinant monoclonal antibodies secreted into the culture supernatant overnight after induction were tested for binding to biotinylated TRBC1 and TRBC2 immobilized on neutravidin-coated Nunc Maxisorp™ 96-well plates. Of the 564 clones sorted from TRBC1 selections, 255 clones were found to be specific for TRBC1 (>10,000 TRF units for TRBC1 and <1,000 TRF units for TRBC2). 138 TRBC2-specific binders (>10,000 TRF units for TRBC2 and <2,000 TRF units for TRBC1) were identified from the 564 clones sorted from TRBC2 selections. Figure 24 shows a representative binding profile from a single 96-well plate obtained from either TRBC1 (Figure 24A) or TRBC2 (Figure 24B) selection. Details of the specific binders obtained using the different selection conditions are summarized in Table 5.

142 and 138 specific binders were selected from the TRBC1 and TRBC2 screens, respectively, for sequence analysis and further characterization. Sequences of the highly selected clones were obtained by Sanger sequencing using the BigDye® terminator v3.1 cycle sequencing kit (Life technologies). DNA sequences were analyzed to determine protein sequence and the CDRs of the VH and VL domains were identified. Analysis of the VH and VL CDR3 regions identified 74 unique TRBC1 clones and 42 unique TRBC2 clones (where uniqueness was defined as any combination of VH CDR3 and VL CDR3 sequences). TRBC1-specific clones and their VH CDR3 and VL CDR3 sequences are summarized in Table 1 above. TRBC2-specific clones and their VH CDR3 and VL CDR3 sequences are summarized in Table 2 above.

Table 3A. Solid-Phase TRBC Screening Details

Solid phase selections Antigen for selection Number of rounds Antigen concentration Immobilization Antigen deselection in round 1 Deselection of antigens in round 2 BSA TRBC1 (Round 1),
OA TRBC1 (Round 2) 2 10 mcg/ml Straight BSA (100 µg/ml), TRBC2 (30 µM) TRBC2 (30 μM) BSA TRBC2 (Round 1),
OA TRBC2 (Round 2) 2 10 mcg/ml Straight BSA (100 µg/ml), TRBC1 (30 µM) TRBC1 (30 μM) Bio-TRBC1 (Round 1 and 2) 2 3 mcg/ml Streptavidin beads (Round 1), Neutravidin
(Round 2) Streptavidin beads, TRBC2 (30 µg/ml) Neutravidin Granules, TRBC2 (30 mcg/ml) Bio-TRBC2 (Round 1 and 2) 2 3 mcg/ml Streptavidin beads (Round 1), Neutravidin
(Round 2) Streptavidin beads, TRBC1 (30 µg/ml) Neutravidin Granules, TRBC1 (30 mcg/ml)

Table 3B Liquid Phase TRBC Screening Details

Liquid phase sampling Antigen for selection Number of rounds Antigen concentration Antigen deselection in round 1 Deselection of antigens in round 2 Deselection of antigens in round 3 Bio-TRBC1 (Round 1,2 and 3) 3 500 nM Streptavidin beads, TRBC2 (5 μM) Streptavidin beads, TRBC2
(5 μM) Neutravidin beads, TRBC2 (5 μM) Bio-TRBC2 (Round 1,2 and 3) 3 500 nM Streptavidin beads, TRBC1 (5 μM) Streptavidin beads, TRBC1
(5 μM) Neutravidin beads, TRBC1 (5 μM)

Table 4: Quantities from selections

Selection type Number of rounds Antigen Number of plaque-forming units (Round 1) Plaque Forming Number (Round 2) Plaque Forming Number (Round 3) Solid phase 2 BSA/OA TRBC1 1.0×10 ⁴ 6.0×10 ⁵ N.D. Solid phase 2 BSA/OA TRBC2 1.5×10 ³ 2.2×10 ⁶ N.D. Solid phase 2 Bio-TRBC1 5.0×10 ³ 2.0×10 ⁵ N.D. Solid phase 2 Bio-TRBC2 3.0×10 ³ 1.0×10 ⁴ N.D. Liquid phase 3 Bio-TRBC1 1.5×10 ⁶ >10 ⁸ >10 ⁸ Liquid phase 3 Bio-TRBC2 2.7×10 ⁵ >10 ⁸ >10 ⁸

Table 5: Monoclonal screening details

Selection number Selection type Antigen for selection Intermediate selection result Number of clones selected Number of specific binding agents 262 Solid phase, indirect immobilization TRBC1 Round 2 186 93 263 Solid phase, indirect immobilization TRBC2 Round 2 186 68 264 Liquid phase TRBC1 Round 2 186 83 265 Liquid phase TRBC2 Round 2 186 29 266 Liquid phase TRBC1 Round 3 94 47 267 Liquid phase TRBC2 Round 3 94 33 268 Solid phase, direct immobilization (BSA/OA) TRBC1 Round 2 94 32 269 Solid phase, direct immobilization (BSA/OA) TRBC2 Round 2 94 9

Example 13 - Production of polyclonal antibody to TRBC by immunization of rabbits with peptides

In order to obtain antibodies that differentiate between TRBC2 and TRBC1, two peptides were synthesized that cover the major region of differentiation between the two TRBC isoforms and used to immunize rabbits. The following peptide sequences were used:

TRBC1: VLEDLNKVFPPEVAVC (SEQ ID No. 38)

TRBC2: VLEDLKNVFPPEVAVC (SEQ ID No. 39).

Fifteen mg of TRBC1 and TRBC2 peptides were synthesized. Keyhole limpet hemocyanin (KLH) was conjugated to TRBC1 and TRBC2 peptides via the C-terminal cysteines present on the peptides. Two New England rabbits were immunized for each peptide; a total of three times with KLH-conjugated TRBC1 or TRBC2 peptide. After the third immunization, the rabbits were sacrificed and exsanguinated, and the serum was collected for purification. Crude serum from the rabbits was passed through a column of cross-linked agarose resin beads along with the peptide used for immunization to collect antibodies specific for common segments and the TRBC isoform-specific epitope of the peptide. The initially purified supernatant was then further purified using an immobilized alternative peptide column to remove antibodies specific to common segments of the peptide.

ELISA protocol

Coating antigen(s):

A: TRBC1 peptide

B: TRBC2 peptide

Coating concentration: 4 µg/ml, 100 µl/well

Coating buffer: Phosphate buffered saline, pH7.4

Secondary antibody: Goat anti-rabbit IgG (H&L) conjugated with peroxidase

The results are shown in Figures 25 and 26. By this method, it is possible to create a polyclonal serum containing antibodies specific to TRBC1 or TRBC2.

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245 250

<210> 20

<211> 244

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_01_D03

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Lys Gln Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Ala Ser Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser

130 135 140

Leu Ala Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

145 150 155 160

Val Gly Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

165 170 175

Ser Leu Leu Ile Tyr Asp Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Ser Glu Asp Ile Ala Val Tyr Tyr Cys Gln Gln Tyr His

210 215 220

Arg Trp Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

225 230 235 240

Th Ala Ala Ala

<210> 21

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_01_F06

<400> 21

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asp Asp Gly Ala Met Arg Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Ala Ser Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val

130 135 140

Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val

145 150 155 160

Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

180 185 190

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr

210 215 220

Cys Gln Gln Tyr Tyr Asp Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys

225 230 235 240

Val Asp Ile Lys Arg Thr Ala Ala Ala

245

<210> 22

<211> 244

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_01_F02

<400> 22

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Pro Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Gly Tyr Ser Tyr Ala Asp Tyr Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Ala Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser

130 135 140

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly

145 150 155 160

Ile Arg Asn Asp Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

165 170 175

Lys Arg Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

180 185 190

Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser

195 200 205

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn

210 215 220

Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg

225 230 235 240

Th Ala Ala Ala

<210> 23

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_E05

<400> 23

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Thr Arg Ser Ser Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Ala Ser Asn Phe Met Leu Thr Gln Pro His Ser Val Ser

130 135 140

Glu Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly

145 150 155 160

Ser Ile Ala Ser Lys Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser

165 170 175

Ser Pro Thr Thr Val Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val

180 185 190

Pro Asp Arg Phe Ser Gly Ser Ile Asp Thr Ser Ser Asn Ser Ala Ser

195 200 205

Leu Thr Ile Ser Gly Leu Arg Thr Glu Asp Glu Ala Asp Tyr Tyr Cys

210 215 220

His Ser Tyr Asp Ser Asn Asn His Ser Val Phe Gly Gly Gly Thr Lys

225 230 235 240

Val Thr Val Leu Gly Gln Pro Ala Ala

245

<210> 24

<211> 246

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_D05

<400> 24

Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Arg Gly Arg Gly Ser Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Ala Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val

130 135 140

Ser Val Ser Pro Gly Gln Thr Ala Thr Ile Ser Cys Ser Gly Asp Gln

145 150 155 160

Leu Gly Gly Lys Tyr Gly His Trp Tyr Gln Lys Lys Pro Gly Gln Ser

165 170 175

Pro Val Leu Val Leu Tyr Gln Asp Arg Lys Arg Pro Ala Gly Ile Pro

180 185 190

Glu Arg Phe Ser Gly Ser Ser Ser Gly Asn Thr Ile Thr Leu Thr Ile

195 200 205

Ser Gly Thr Gln Ala Val Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp

210 215 220

Asp Thr Asn Leu Gly Gly Val Phe Gly Gly Gly Thr Lys Val Thr Val

225 230 235 240

Leu Gly Gln Pro Ala Ala

245

<210> 25

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_H06

<400> 25

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Arg Val Gly Gly Met Asp Val Trp Gly Gln Gly Thr Met

100 105 110

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Ala Ser Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu

130 135 140

Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser

145 150 155 160

Ile Ala Ser Asn Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ser

165 170 175

Pro Thr Thr Val Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val Pro

180 185 190

Asp Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu

195 200 205

Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Gln

210 215 220

Ser Phe Asp Ala Asp Asn Leu His Val Val Phe Gly Gly Gly Thr Lys

225 230 235 240

Leu Thr Val Leu Gly Gln Pro Ala Ala

245

<210> 26

<211> 248

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_C12

<400> 26

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Thr Gly Pro Ile Asp Tyr Trp Gly Gln Gly Thr Met Val

100 105 110

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Ala Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val

130 135 140

Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu

145 150 155 160

Leu His Ser Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro

165 170 175

Gly Gln Pro Pro Gln Leu Leu Val Tyr Glu Val Ser Asn Arg Phe Ser

180 185 190

Gly Val Pro Asp Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

195 200 205

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

210 215 220

Met Gln Gly Ile Gln Leu Pro Pro Thr Phe Gly Gly Gly Thr Lys Val

225 230 235 240

Asp Ile Lys Arg Thr Ala Ala Ala

245

<210> 27

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_G02

<400> 27

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Ile Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Val Trp Asn Ser Gly Ser Tyr Leu Gly Phe Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Ala Ser Asp Ile Gln Met Thr Gln Ser

130 135 140

Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys

145 150 155 160

Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys

165 170 175

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln

180 185 190

Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

195 200 205

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

210 215 220

Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg Thr Ala Ala Ala

245

<210> 28

<211> 251

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_D04

<400> 28

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Phe Thr Val Pro Gly Gly Ala Phe Asp Ile Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Ala Ser Asn Phe Met Leu Thr Gln Pro His

130 135 140

Ser Val Ser Asp Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr Arg

145 150 155 160

Ser Ser Gly Arg Ile Gly Ser Asn Phe Val Gln Trp Tyr Gln Gln Arg

165 170 175

Pro Gly Ser Ser Pro Thr Thr Val Ile Tyr Glu Asp Asp Gln Arg Pro

180 185 190

Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn

195 200 205

Ser Ala Ser Leu Thr Ile Ser Gly Leu Thr Thr Ala Asp Glu Ala Gly

210 215 220

Tyr Tyr Cys Gln Ser Tyr Asp Ala Ser Asn Val Ile Phe Gly Gly Gly

225 230 235 240

Thr Lys Leu Thr Val Leu Gly Gln Pro Ala Ala

245 250

<210> 29

<211> 247

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_F10

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Gly Glu Arg Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Ala Ser Gln Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser

130 135 140

Gly Ser Pro Gly Gln Ser Val Thr Ile Ser Cys Thr Gly Thr Ser Thr

145 150 155 160

Asp Val Gly Ala Phe His Phe Val Ser Trp Tyr Gln His Thr Pro Gly

165 170 175

Lys Ala Pro Lys Leu Leu Ile Ser Glu Val Arg Lys Arg Ala Ser Gly

180 185 190

Val Pro Asp Arg Phe Ser Gly Ser Arg Ser Gly Asn Thr Ala Ser Leu

195 200 205

Thr Val Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Phe Cys Ser

210 215 220

Ala Tyr Thr Gly Ser Asn Tyr Val Phe Gly Ser Gly Thr Lys Leu Thr

225 230 235 240

Val Leu Gly Gln Pro Ala Ala

245

<210> 30

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_G09

<400> 30

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Gln Trp Leu Ala Asn Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ala Ser Ser Tyr Glu

130 135 140

Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln Thr Ala Arg

145 150 155 160

Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val His Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Arg Asp Asn

180 185 190

Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly

195 200 205

Asn Thr Ala Thr Leu Thr Ile Ser Lys Ala Gln Ala Gly Asp Glu Ala

210 215 220

Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Trp Val Phe Gly Gly

225 230 235 240

Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Ala Ala

245 250

<210> 31

<211> 252

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_F09

<400> 31

Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Asn Arg Gly Gly Ser Tyr Lys Ser Val Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Ala Ser Asn Phe Met Leu Thr Gln Pro

130 135 140

Gln Ser Val Ser Glu Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr

145 150 155 160

Arg Ser Ser Gly Asn Phe Ala Ser Lys Tyr Val Gln Trp Tyr Gln Gln

165 170 175

Arg Pro Gly Ser Ser Pro Thr Thr Val Ile Tyr Glu Asn Tyr Gln Arg

180 185 190

Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser

195 200 205

Asn Ser Ala Thr Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala

210 215 220

Asp Tyr Tyr Cys Gln Ser Tyr Asp Glu Val Ser Val Val Phe Gly Gly

225 230 235 240

Gly Thr Gln Leu Thr Val Leu Gly Gln Pro Ala Ala

245 250

<210> 32

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> scFv CP_03_D09

<400> 32

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Glu Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Ser Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Ala Ser Asn Phe Met Leu Thr Gln Pro Leu Ser Val Ser

130 135 140

Glu Ser Pro Gly Lys Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly

145 150 155 160

Ser Ile Ala Ser Asn Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser

165 170 175

Ala Pro Thr Thr Val Ile Tyr Glu Asp Asn Gln Arg Pro Ser Gly Val

180 185 190

Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser

195 200 205

Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp Glu Ala Asp Tyr Tyr Cys

210 215 220

Gln Ser Tyr Asn Ser Ser Asn His Trp Val Phe Gly Gly Gly Thr Lys

225 230 235 240

Val Thr Val Leu Gly Gln Pro Ala Ala

245

<210> 33

<211> 537

<212> PRT

<213> Artificial Sequence

<220>

<223> Chimeric antigen receptor (CAR), JOVI-1 CAR with CD8

stalk spacer

<400> 33

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Val Trp Ile Pro

1 5 10 15

Gly Ser Thr Gly Glu Val Arg Leu Gln Gln Ser Gly Pro Asp Leu Ile

20 25 30

Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Gly Tyr Val Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

50 55 60

Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser

65 70 75 80

Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser

85 90 95

Thr Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr

115 120 125

Arg Phe Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp

165 170 175

Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn

180 185 190

Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

195 200 205

Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

225 230 235 240

Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr

245 250 255

His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

260 265 270

Ser Asp Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

275 280 285

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

290 295 300

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

305 310 315 320

Ile Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

325 330 335

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg

340 345 350

Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro

355 360 365

Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe

370 375 380

Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys

385 390 395 400

Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala

405 410 415

Asp Ala His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser

420 425 430

Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu

435 440 445

Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg

450 455 460

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

465 470 475 480

Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr

485 490 495

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

500 505 510

Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala

515 520 525

Leu His Met Gln Ala Leu Pro Pro Arg

530 535

<210> 34

<211> 727

<212> PRT

<213> Artificial Sequence

<220>

<223> JOVI-1 CAR with H-CH2-CH3pvaa spacer

<400> 34

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Val Trp Ile Pro

1 5 10 15

Gly Ser Thr Gly Glu Val Arg Leu Gln Gln Ser Gly Pro Asp Leu Ile

20 25 30

Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Gly Tyr Val Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

50 55 60

Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser

65 70 75 80

Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser

85 90 95

Thr Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr

115 120 125

Arg Phe Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp

165 170 175

Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn

180 185 190

Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

195 200 205

Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

225 230 235 240

Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr

245 250 255

His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

260 265 270

Ser Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro

275 280 285

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

290 295 300

Pro Lys Pro Lys Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr

305 310 315 320

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

325 330 335

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

340 345 350

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

355 360 365

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

370 375 380

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

385 390 395 400

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

405 410 415

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

420 425 430

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

435 440 445

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

450 455 460

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

465 470 475 480

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

485 490 495

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Lys Asp Pro Lys Phe

500 505 510

Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu

515 520 525

Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg

530 535 540

Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro

545 550 555 560

Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala

565 570 575

Tyr Arg Ser Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro

580 585 590

Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala

595 600 605

His Ser Thr Leu Ala Lys Ile Arg Val Lys Phe Ser Arg Ser Ala Asp

610 615 620

Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn

625 630 635 640

Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg

645 650 655

Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly

660 665 670

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

675 680 685

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

690 695 700

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

705 710 715 720

Met Gln Ala Leu Pro Pro Arg

725

<210> 35

<211> 511

<212> PRT

<213> Artificial Sequence

<220>

<223> JOVI-1 CAR with IgG1 hinge spacer

<400> 35

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Val Trp Ile Pro

1 5 10 15

Gly Ser Thr Gly Glu Val Arg Leu Gln Gln Ser Gly Pro Asp Leu Ile

20 25 30

Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Gly Tyr Val Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

50 55 60

Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln Ser

65 70 75 80

Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser

85 90 95

Thr Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala Tyr

115 120 125

Arg Phe Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

145 150 155 160

Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp

165 170 175

Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Val His Ser Asn

180 185 190

Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

195 200 205

Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Pro Gly Val Pro Asp

210 215 220

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser

225 230 235 240

Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr

245 250 255

His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

260 265 270

Ser Asp Pro Ala Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro

275 280 285

Pro Cys Pro Lys Asp Pro Lys Phe Trp Val Leu Val Val Val Gly Gly

290 295 300

Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe

305 310 315 320

Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn

325 330 335

Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr

340 345 350

Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Asp Gln Arg Leu

355 360 365

Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro

370 375 380

Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Arg

385 390 395 400

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

405 410 415

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

420 425 430

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

435 440 445

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

450 455 460

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

465 470 475 480

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

485 490 495

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

500 505 510

<210> 36

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic peptide for TRBC2

<400> 36

Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val

1 5 10 15

<210> 37

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic peptide for TRBC1

<400> 37

Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val

1 5 10 15

<210> 38

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic peptide for TRBC1

<400> 38

Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Cys

1 5 10 15

<210> 39

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic peptide for TRBC2

<400> 39

Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Cys

1 5 10 15

<210> 40

<211> 175

<212> PRT

<213> Homo sapiens

<400> 40

Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser

1 5 10 15

Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala

20 25 30

Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

35 40 45

Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu

50 55 60

Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg

65 70 75 80

Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln

85 90 95

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg

100 105 110

Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala

115 120 125

Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala

130 135 140

Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val

145 150 155 160

Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

<210> 41

<211> 175

<212> PRT

<213> Homo sapiens

<400> 41

Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser

1 5 10 15

Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala

20 25 30

Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly

35 40 45

Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu

50 55 60

Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg

65 70 75 80

Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln

85 90 95

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg

100 105 110

Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala

115 120 125

Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala

130 135 140

Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val

145 150 155 160

Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp

165 170 175

<210> 42

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_E09

<400> 42

Ala His Asn Ser Ser Ser Trp Ser Phe

1 5

<210> 43

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D12

<400> 43

Gly Gly Asp Thr Tyr Gly Phe Leu

1 5

<210> 44

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D10

<400> 44

Gly Gly Gly Ser Phe Gly Ala Phe

1 5

<210> 45

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_C08

<400> 45

Gly Tyr Ser Ser Ser Trp Tyr Leu

1 5

<210> 46

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_C11

<400> 46

Gly Gly Ala Gly

1

<210> 47

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F03

<220>

<221> misc_feature

<222> (3)..(3)

<223> Xaa can be any naturally occurring amino acid

<400> 47

Gly Tyr Xaa Ala Ser Ser Trp Ser Gln

1 5

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_E07

<400> 48

Asp Leu Gly Gly Ser Gly Gly Ala Phe

1 5

<210> 49

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D03

<400> 49

AsnLysGlnTyrGlyMet

1 5

<210> 50

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F06

<400> 50

Asp Asp Gly Ala Met

1 5

<210> 51

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F02

<400> 51

Ala Gly Tyr Ser Tyr Ala

1 5

<210> 52

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_C03

<400> 52

Gly Gly Arg Tyr Ser Ser Asn Tyr Phe

1 5

<210> 53

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_D10

<400> 53

Val Gly Glu Gly Ser Ala Met

1 5

<210> 54

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_B01

<400> 54

Val Ser Ser His Tyr Asp Ser Ser Gly Tyr Tyr Ala Gly Gly Phe

1 5 10 15

<210> 55

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_D02

<400> 55

Gly Arg Asp Ser Ser Ser Trp Ser Pro

1 5

<210> 56

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A02

<400> 56

Val Thr Thr Tyr Ser Gly Leu Asp Phe

1 5

<210> 57

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_D04

<400> 57

Lys Gly Ala Val Val Val Pro Gly Ala Leu

1 5 10

<210> 58

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_E10

<400> 58

Asn Ser Leu Tyr Gly Gly Asn Ser Ala

1 5

<210> 59

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_H08

<400> 59

Asp Gly Gly Gly Gly Arg Phe

1 5

<210> 60

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F11

<400> 60

Gly Gly Gly Ala Leu Gly Arg Gly Met

1 5

<210> 61

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F09

<400> 61

Leu Leu Arg Ser Gly Gly Gln Ser Tyr Ala Phe

1 5 10

<210> 62

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_D05

<400> 62

Gly Tyr Ser Ser Ser Trp Ser Phe

1 5

<210> 63

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A09

<400> 63

Ala Gly Ser Ser Gly Trp Thr Leu

1 5

<210> 64

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_D03

<400> 64

Asp Lys Gly Trp Gly Phe

1 5

<210> 65

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_C11

<400> 65

Leu Gly Val Val Arg Gly Val Met Lys Gly Phe

1 5 10

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_H10

<400> 66

Ser Ser Tyr Ser Ser Ser Trp Gly Met

1 5

<210> 67

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_C04

<400> 67

Ala Asn Ser Trp Ser Ala Gly Gly Met

1 5

<210> 68

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G03

<400> 68

Glu Arg Gly Arg Gly Tyr Ser Tyr Met

1 5

<210> 69

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G06

<400> 69

Val Ala Arg Gly Ile His Asp Ala Phe

1 5

<210> 70

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D06

<400> 70

Arg His Gly Met

1

<210> 71

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_B03

<400> 71

Phe Asp Ser Ser Gly Tyr Tyr Tyr

1 5

<210> 72

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A12

<400> 72

Asp Leu Val Thr Thr Gly Ala Phe

1 5

<210> 73

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_H03

<400> 73

Ala Ile Arg Val Ser Gly Thr Pro Glu Asn Gly Phe

1 5 10

<210> 74

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G08

<400> 74

Val Arg Ile Thr His Gly Met

1 5

<210> 75

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_A06

<400> 75

Gly Lys Leu Ala Phe

1 5

<210> 76

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A04

<400> 76

Asn Gly Asp Ser Ser Gly Tyr His Thr Ser Pro Asn Trp Tyr Phe

1 5 10 15

<210> 77

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_E08

<400> 77

Val Ser Thr Asp Ser Ser Ser Met

1 5

<210> 78

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_A08

<400> 78

Thr Ser Gln Asp Pro Gly Ala Phe

1 5

<210> 79

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D01

<400> 79

Ala Glu Ser Gly Val Tyr Ser Ser Asn Gly Met

1 5 10

<210> 80

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A07

<400> 80

Val Asp Arg Val Arg Ser Gly Met

1 5

<210> 81

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_B08

<400> 81

Ile Gly Gln Tyr Cys Ser Ser Thr Ser Cys Tyr Met

1 5 10

<210> 82

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D09

<400> 82

Asp Leu Gly Gly Ser Gly Gly Ala Phe

1 5

<210> 83

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G07

<400> 83

Asp Ser Asp Ala Gly Tyr Phe

1 5

<210> 84

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_A05

<400> 84

Ala Ser Ile Val Ala Ser Gly Ala Phe

1 5

<210> 85

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A08

<400> 85

Ala Gly Gly Ser Asn Ala Phe

1 5

<210> 86

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_D07

<400> 86

Val Ser Thr Asp Ser Tyr Gly Arg Gln Asn Trp Phe

1 5 10

<210> 87

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_C04

<400> 87

Gln Tyr Thr Ser Gly Arg Leu Ala Tyr Tyr Tyr His Tyr Met

1 5 10

<210> 88

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_A07

<400> 88

Gly Ile Arg Gly Ala Phe

1 5

<210> 89

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_H02

<400> 89

Val Gly Tyr Ser Thr Thr Gln Leu

1 5

<210> 90

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F10

<400> 90

Met Ala Gly Ser Tyr Tyr Ala Phe

1 5

<210> 91

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_C10

<400> 91

Val Gly Asp Tyr Tyr Asp Ser Ser Gly Tyr Leu Asp Trp Tyr Phe

1 5 10 15

<210> 92

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_B05

<400> 92

Gly Ser Asp Thr Thr Ser Phe Val Ser

1 5

<210> 93

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G04

<400> 93

Ala Gly His Tyr Tyr Tyr Tyr Met

1 5

<210> 94

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F08

<400> 94

Val Thr Gly Tyr Pro Asp Tyr Tyr Asp Ser Ser Gly Phe

1 5 10

<210> 95

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G05

<400> 95

Val Glu Gly Gly Pro Pro Tyr Tyr Phe

1 5

<210> 96

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A03

<400> 96

Asn Gly Leu Asp Asn Tyr Gly Met

1 5

<210> 97

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_B09

<400> 97

Leu Gly Thr Thr Lys Arg Ala Phe

1 5

<210> 98

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_A10

<400> 98

Val Tyr Val Asp His Glu Gly Met

1 5

<210> 99

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_H04

<400> 99

Trp Ser Gly Ser Gly Phe

1 5

<210> 100

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_B04

<400> 100

Asp Phe Gly Trp Gly Gly Ala Phe

1 5

<210> 101

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A05

<400> 101

Val Val Gly Gly Thr Gln His

1 5

<210> 102

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_F07

<400> 102

Asn Trp Leu Leu Tyr Tyr Gly Asp Pro Gln Gln Asn Ala Phe

1 5 10

<210> 103

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_H01

<400> 103

Leu Tyr Phe Asp Trp Phe Ala Asp Ser Gln Asn Ala Phe

1 5 10

<210> 104

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G10

<400> 104

Val Gly Tyr Gln Pro Leu Leu Tyr Ala Asp Tyr Tyr Phe

1 5 10

<210> 105

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G11

<400> 105

Gly Ala Met Gly Leu

1 5

<210> 106

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G01

<400> 106

Val Tyr Tyr Leu Ser Gly Val His Ala Phe

1 5 10

<210> 107

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_A12

<400> 107

Thr Glu Arg Trp Leu Gln Phe

1 5

<210> 108

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_H05

<400> 108

Asn Gly Asp Tyr Ala Phe

1 5

<210> 109

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_B07

<400> 109

Ala Ser Arg Tyr Ser Gly Ser Tyr His Phe

1 5 10

<210> 110

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_G09

<400> 110

His Gly Ser Gln Gly Gly Phe

1 5

<210> 111

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_C02

<400> 111

Val Gly Tyr Met Gly Gly Met

1 5

<210> 112

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D05

<400> 112

Asn Thr Pro Gly Ile Ala Ala Ala Gly Pro

1 5 10

<210> 113

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_01_D08

<400> 113

Val Gly Thr Thr Val Thr Ser Phe

1 5

<210> 114

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_A11

<400> 114

Val Gly Gly Pro Leu Asn Asp Ala Phe

1 5

<210> 115

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_D08

<400> 115

His Ser Ser Gly Gly Ala Phe

1 5

<210> 116

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_E09

<400> 116

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 117

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D12

<400> 117

Gln Gln Phe Asn Ala Tyr Pro

1 5

<210> 118

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D10

<400> 118

Gln Gln Tyr Asn Ser Tyr Pro

1 5

<210> 119

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_C08

<400> 119

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 120

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_C11

<400> 120

Gln Ser His Asp Ser Ser Asn

1 5

<210> 121

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F03

<400> 121

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 122

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_E07

<400> 122

Met Gln Ser Ile Gln Leu

1 5

<210> 123

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D03

<400> 123

Gln Gln Tyr His Arg Trp Pro

1 5

<210> 124

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F06

<400> 124

Gln Gln Tyr Tyr Asp Ser Pro

1 5

<210> 125

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F02

<400> 125

Leu Gln His Asn Ser Tyr Pro

1 5

<210> 126

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_C03

<400> 126

Gln Gln Tyr Phe Gly Thr

1 5

<210> 127

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_D10

<400> 127

Gln Gln Tyr Asn Asp Trp Pro

1 5

<210> 128

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_B01

<400> 128

Gln Ser Phe Asp Thr Asn Ser Leu

1 5

<210> 129

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_D02

<400> 129

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 130

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A02

<400> 130

Ser Ser Tyr Thr Ser Ser Ser Thr

1 5

<210> 131

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_D04

<400> 131

Gln Gln Tyr Asn Ser Tyr Pro

1 5

<210> 132

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_E10

<400> 132

Gln Gln Thr Phe Thr Thr Pro

1 5

<210> 133

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_H08

<400> 133

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 134

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F11

<400> 134

Gln Ser Tyr Asp Thr Asn Asn

1 5

<210> 135

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F09

<400> 135

Gln Gln Ser Tyr Ser Thr Pro

1 5

<210> 136

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_D05

<400> 136

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 137

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A09

<400> 137

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 138

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_D03

<400> 138

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 139

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_C11

<400> 139

Gln Ser Tyr Asp Ser Ser Asn

1 5

<210> 140

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_H10

<400> 140

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 141

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_C04

<400> 141

Gln Gln Tyr Asp Asp Leu Pro

1 5

<210> 142

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G03

<400> 142

Met Gln Arg Ile Glu Phe Pro

1 5

<210> 143

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G06

<400> 143

Gln Ser Tyr Asp Asn Thr Arg His

1 5

<210> 144

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D06

<400> 144

Gln Ser Tyr Asp Ser Ser Asn

1 5

<210> 145

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_B03

<400> 145

Gln Ser Tyr Asp Ser Ser Asn

1 5

<210> 146

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A12

<400> 146

Gln Gln His Asn Asp Trp Pro

1 5

<210> 147

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_H03

<400> 147

Gln Ser Tyr His Ser Ser Asn Leu

1 5

<210> 148

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G08

<400> 148

Met Gln Ala Thr His Phe Pro

1 5

<210> 149

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_A06

<400> 149

Gln Gln Tyr Tyr Ser Thr Pro

1 5

<210> 150

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A04

<400> 150

Gln Ser Tyr Asp Asp Ser Asn Tyr

1 5

<210> 151

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_E08

<400> 151

Ser Ser Tyr Ala Gly Ser Asn Thr Leu

1 5

<210> 152

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_A08

<400> 152

Gln Gln Ala Asn Ser Phe Pro

1 5

<210> 153

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D01

<400> 153

Gln Ser Tyr Asp Ser Ser Ile

1 5

<210> 154

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A07

<400> 154

Gln Ser Tyr Asp Ser Ile His

1 5

<210> 155

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_B08

<400> 155

Gln Ser Tyr Asp Ser Ser Thr His

1 5

<210> 156

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D09

<400> 156

Gln Gln Tyr Asp Asn Leu Pro

1 5

<210> 157

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G07

<400> 157

Gln Ser Phe Thr Ser Ser Thr Leu

1 5

<210> 158

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_A05

<400> 158

Gln Gln Tyr Asn Lys Trp Pro

1 5

<210> 159

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A08

<400> 159

Gln Ser Tyr Asp Asp Ser Asn Tyr

1 5

<210> 160

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_D07

<400> 160

Gln Ser Tyr Asp Ser Ser Asn His

1 5

<210> 161

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_C04

<400> 161

Gln Gln Ser Tyr Ser Thr Pro

1 5

<210> 162

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_A07

<400> 162

Asn Ser Arg Asp Ser Ser Gly Asn Pro Asn

1 5 10

<210> 163

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_H02

<400> 163

Gln Ser Tyr Asp Ser Ser Asn Leu

1 5

<210> 164

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F10

<400> 164

Gln Ser Tyr Asp Ser Ser Asn His

1 5

<210> 165

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_C10

<400> 165

Ala Val Trp Asp Asp Arg Leu Asn Gly

1 5

<210> 166

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_B05

<400> 166

Gln Gln Tyr Asp Ser Tyr Ser

1 5

<210> 167

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G04

<400> 167

Gln Ser Ala Asp Ser Ser Gly Thr Asn

1 5

<210> 168

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F08

<400> 168

Gln Ser Tyr Asp Ser Ser Asn His

1 5

<210> 169

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G05

<400> 169

Gln Ser Tyr Asp Thr Arg Asn Gln

1 5

<210> 170

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A03

<400> 170

Gln Gln Tyr Tyr Ser Thr Pro

1 5

<210> 171

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_B09

<400> 171

Gln Gln Ser Tyr Ser Thr

1 5

<210> 172

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_A10

<400> 172

Ala Ala Trp Asp Asp Ser Leu Phe

1 5

<210> 173

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_H04

<400> 173

Met Gln Arg Ile Glu Phe Pro

1 5

<210> 174

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_B04

<400> 174

Gln Gln Tyr Tyr Asn Thr Pro

1 5

<210> 175

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A05

<400> 175

Gln Ser Tyr Asp Ser Ser Ile

1 5

<210> 176

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_F07

<400> 176

Gln Ser Tyr Asp Ser Thr Asn Leu

1 5

<210> 177

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_H01

<400> 177

Gln Gln Ser Tyr Ser Thr Pro

1 5

<210> 178

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G10

<400> 178

Asn Ser Arg Asp Ser Ser Gly Asn His

1 5

<210> 179

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G11

<400> 179

Gln Gln Ser Tyr Ser Thr Pro

1 5

<210> 180

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G01

<220>

<221> misc_feature

<222> (9)..(9)

<223> Xaa can be any naturally occurring amino acid

<400> 180

Asp Ser Arg Asp Thr Arg Val Asn Xaa

1 5

<210> 181

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_A12

<400> 181

Gln Gln Tyr Asp Asn Leu

1 5

<210> 182

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_H05

<400> 182

Met Gln Ala Leu Gln Thr Pro

1 5

<210> 183

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_B07

<400> 183

Gln Ser Tyr Asp Ser Ser Asn

1 5

<210> 184

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_G09

<400> 184

Ser Ser Tyr Thr Ser Ser Ser Thr

1 5

<210> 185

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_C02

<400> 185

Met Gln Ala Leu Gln Thr Pro Pro

1 5

<210> 186

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D05

<400> 186

Gln Ser Tyr Asp Ser Thr Asn His

1 5

<210> 187

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_01_D08

<400> 187

Gln Ser Tyr Asp Ser Ala Asn Leu

1 5

<210> 188

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_A11

<400> 188

Gln Ser Phe Asp Glu Asn Ile Ser

1 5

<210> 189

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_D08

<400> 189

His Gln Ser Ala Thr Ser Pro

1 5

<210> 190

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_E05

<400> 190

Thr Arg Ser Ser Gly Ala Phe

1 5

<210> 191

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_D05

<400> 191

Pro Arg Gly Arg Gly Ser Ala Phe

1 5

<210> 192

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_H06

<400> 192

Ala Arg Val Gly Gly Met

1 5

<210> 193

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_C12

<400> 193

Asp Thr Gly Pro Ile

1 5

<210> 194

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_G02

<400> 194

Gly Val Trp Asn Ser Gly Ser Tyr Leu Gly Phe

1 5 10

<210> 195

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_D04

<400> 195

Gly Gly Phe Thr Val Pro Gly Gly Ala Phe

1 5 10

<210> 196

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_F10

<400> 196

Phe Gly Glu Arg Tyr Ala Phe

1 5

<210> 197

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_G09

<400> 197

Asp Gln Trp Leu Ala Asn Tyr Tyr Tyr Tyr Gly Met

1 5 10

<210> 198

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_F09

<400> 198

Asn Arg Gly Gly Ser Tyr Lys Ser Val Gly Met

1 5 10

<210> 199

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_D09

<400> 199

Val Ser Ser Tyr Tyr Gly Met

1 5

<210> 200

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_F02

<400> 200

Ala Pro Ala Ser Ser Ala His

1 5

<210> 201

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_02_E03

<400> 201

Gln Arg Gly Tyr Tyr Tyr Gly Met

1 5

<210> 202

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_H07

<400> 202

Ser Ser Val Ala Ala Gly Ala Phe

1 5

<210> 203

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_C02

<400> 203

Leu Ser Gly Arg Gly Leu Gly Phe

1 5

<210> 204

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_E09

<400> 204

Asp His Tyr Phe

1

<210> 205

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_D08

<400> 205

Ser Gly Arg Arg Val Thr Ala Ile

1 5

<210> 206

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_E11

<400> 206

Met Gly Arg Tyr Ser Ser Ser Trp

1 5

<210> 207

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_B05

<400> 207

His Ser Arg Phe Gly Pro Ala Phe

1 5

<210> 208

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_H02

<400> 208

Asp Arg Glu Ala Phe

1 5

<210> 209

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_D02

<400> 209

Leu Arg Gly Arg Tyr Ser Tyr Gly Tyr Ser Asp Ala Phe

1 5 10

<210> 210

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_E01

<400> 210

Leu Leu Asn Ala Val Thr Tyr Ala Phe

1 5

<210> 211

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_C11

<400> 211

Ile Gly Val Ile Gly Gly Phe

1 5

<210> 212

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_B12

<400> 212

Ile Glu Tyr Ser Ser Ser Ser Pro Tyr Phe

1 5 10

<210> 213

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_E03

<400> 213

Asp Leu Leu Pro Thr Thr Val Thr Thr Thr Gly Ala Phe

1 5 10

<210> 214

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_F01

<400> 214

Asp Ser Gly Ser Tyr Ser

1 5

<210> 215

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_C01

<400> 215

Ala Ser Tyr Pro Tyr Tyr Tyr Tyr Tyr Tyr Gly Met

1 5 10

<210> 216

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_G07

<400> 216

Ala Leu Gly His Phe

1 5

<210> 217

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_E02

<400> 217

Phe Thr Thr Gly Ser Ala Leu

1 5

<210> 218

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_C07

<400> 218

Asp Ala Ser Gly Tyr

1 5

<210> 219

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_H04

<400> 219

Asp Leu Gly Thr Tyr Tyr Tyr Gly Ser Gly Asp

1 5 10

<210> 220

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_E06

<400> 220

Val Gly Glu Leu Leu Gly Ala Phe

1 5

<210> 221

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_E05

<400> 221

His Ser Tyr Asp Ser Asn Asn His

1 5

<210> 222

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_G11

<400> 222

His Ser Gly Val Gly Gly Leu Ala Phe

1 5

<210> 223

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_G01

<400> 223

Gly Gly Ser Ile Ala Ala Ala Leu Ala Phe

1 5 10

<210> 224

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_H01

<400> 224

Val Glu Tyr Ser Arg Asn Gly Met

1 5

<210> 225

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_F11

<400> 225

Gly Arg Tyr Asn

1

<210> 226

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_C06

<400> 226

Leu Asp Tyr Tyr Tyr Gly Met

1 5

<210> 227

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_D03

<400> 227

Gly Gly Leu Ser Ser Ala Phe

1 5

<210> 228

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_G05

<400> 228

Tyr Gly Gly Gly Leu

1 5

<210> 229

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_G12

<400> 229

Pro Asp His Leu Thr Val Phe

1 5

<210> 230

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_C10

<400> 230

Val Gly Tyr Tyr Gly Met

1 5

<210> 231

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy CDR3 Clone CP_03_F04

<400> 231

Tyr Glu Gly Tyr Ala Gly Phe

1 5

<210> 232

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_D05

<400> 232

Gln Ala Trp Asp Thr Asn Leu Gly

1 5

<210> 233

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_H06

<400> 233

Gln Ser Phe Asp Ala Asp Asn Leu His

1 5

<210> 234

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_C12

<400> 234

Met Gln Gly Ile Gln Leu Pro

1 5

<210> 235

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_G02

<400> 235

Gln Gln Ser Tyr Ser Thr Pro

1 5

<210> 236

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_D04

<400> 236

Gln Ser Tyr Asp Ala Ser Asn

1 5

<210> 237

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_F10

<400> 237

Ser Ala Tyr Thr Gly Ser Asn

1 5

<210> 238

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_G09

<400> 238

Gln Val Trp Asp Ser Asn Ser

1 5

<210> 239

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_F09

<400> 239

Gln Ser Tyr Asp Glu Val Ser

1 5

<210> 240

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_D09

<400> 240

Gln Ser Tyr Asn Ser Ser Asn His

1 5

<210> 241

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_F02

<400> 241

Gln Ser Tyr Asp Ser Ser His

1 5

<210> 242

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_02_E03

<400> 242

Gln Gln Ser Arg Ser Thr Pro

1 5

<210> 243

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_H07

<400> 243

Ser Ser Tyr Thr Ser Ser Ser Thr

1 5

<210> 244

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_C02

<400> 244

Ser Ser Tyr Ala Gly Ser Ser Asn Leu

1 5

<210> 245

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_E09

<400> 245

Asn Ser Tyr Thr Arg Ser Ser Thr

1 5

<210> 246

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_D08

<400> 246

Gln Ser Tyr Asp Asp Thr Asn

1 5

<210> 247

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_E11

<400> 247

Gln Ala Trp Asp Thr Asn Ile Gly

1 5

<210> 248

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_B05

<400> 248

Ser Ser Tyr Ala Gly Ser Asn Asn

1 5

<210> 249

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_H02

<400> 249

Gln Ala Trp Asp Thr Asn Ile Gly

1 5

<210> 250

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_D02

<400> 250

Ser Ser Tyr Ala Gly Ser Ser Thr

1 5

<210> 251

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_E01

<400> 251

Asn Ser Arg Asp Ser Ser Gly Phe

1 5

<210> 252

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_C11

<400> 252

Ser Ser Tyr Thr Ser Ser Ser

1 5

<210> 253

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_B12

<400> 253

Gln Ser Tyr Asp Ser Asn Asn Arg

1 5

<210> 254

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_E03

<400> 254

Ser Ser Arg Asp Ser Ser Gly Asn His

1 5

<210> 255

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_F01

<400> 255

Gln Ala Trp Asp Thr Asn Ile Gly

1 5

<210> 256

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_C01

<400> 256

Gln Val Trp Asp Ser Ser Thr Ala Asn

1 5

<210> 257

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_G07

<400> 257

Gln Ser Tyr Asp Ser Ser Asn His His

1 5

<210> 258

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_E02

<400> 258

Ser Ser Tyr Ala Gly Asn Ser Asn

1 5

<210> 259

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_C07

<400> 259

Gln Ala Trp Asp Thr Asn Ile Gly

1 5

<210> 260

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_H04

<400> 260

Gly Thr Trp Asp Ser Ser Leu Ser Ala Gly

1 5 10

<210> 261

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_E06

<400> 261

Ser Ser Leu Asp Ser Asn Asp Asn His

1 5

<210> 262

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_H03

<400> 262

Ala Ala Trp Asp Asp Ser Leu Asn Gly

1 5

<210> 263

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_G11

<400> 263

Ser Ser Tyr Ala Gly Ser Ser Thr

1 5

<210> 264

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_G01

<400> 264

His Gln Tyr Asp Val Tyr Pro

1 5

<210> 265

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_H01

<400> 265

Asn Ser Arg Asp Ser Ser Gly Asn His

1 5

<210> 266

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_F11

<400> 266

Gln Ser Tyr Asp Ser Ser Asn

1 5

<210> 267

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_C06

<400> 267

Gln Ser Tyr Asp Ser Ser Asn

1 5

<210> 268

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_D03

<400> 268

Gln Ser Tyr Asp Ser Ser Asn

1 5

<210> 269

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_G05

<400> 269

Ser Ser Tyr Ala Gly Ser Tyr Thr

1 5

<210> 270

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_G12

<400> 270

Ser Ser Tyr Thr Pro Ser Ser

1 5

<210> 271

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_C10

<400> 271

Gln Ala Trp Asp Thr Asn Ile Gly

1 5

<210> 272

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> Light CDR3 Clone CP_03_F04

<400> 272

Gln Ala Trp Asp Thr Asn Ile Gly

1 5

<---

Claims (21)

1. A method for diagnosing T-cell lymphoma or leukemia in a subject, which comprises the step of determining the percentage of T-cells that are TRBC1- or TRBC2-positive out of the total number of T-cells in a sample isolated from said subject, wherein the percentage of TRBC1- or TRBC2-positive T-cells that is greater than about 80% indicates the presence of T-cell lymphoma or leukemia, and wherein the step of determining the percentage of T-cells that are TRBC1- or TRBC2-positive out of the total number of T-cells in said sample isolated from the subject is performed using an agent that comprises an antigen-binding domain that selectively binds to the constant region 1 of TCR beta (TRBC1) or TRBC2. 2. The method according to claim 1, characterized in that said sample is a peripheral blood sample or a biopsy. 3. The method according to any one of paragraphs 1, 2, characterized in that the total number of T cells is detected in said sample or isolated from said sample using an agent that binds to CD3. 4. The method according to any one of claims 1-3, characterized in that said antigen-binding domain selectively binds to TRBC1. 5. The method according to claim 4, characterized in that said antigen-binding domain has a variable heavy chain (VH) and a variable light chain (VL), which contain the following complementarity determining regions (CDR): VH CDR1: SEQ ID NO: 7; VH CDR2: SEQ ID NO: 8; VH CDR3: SEQ ID NO: 9; VL CDR1: SEQ ID NO: 10; VL CDR2: SEQ ID NO: 11; and VL CDR3: SEQ ID NO: 12. 6. The method according to claim 4, characterized in that said antigen-binding domain comprises a variable heavy chain (VH) having the amino acid sequence of SEQ ID NO: 1, and a variable light chain (VL) having the amino acid sequence of SEQ ID NO: 2. 7. The method according to claim 4, characterized in that said antigen-binding domain comprises an scFv with the amino acid sequence SEQ ID NO: 3. 8. The method according to any one of claims 1-3, characterized in that said antigen-binding domain selectively binds to TRBC2. 9. A method for selecting a suitable therapy for treating a subject having a T-cell lymphoma or leukemia, said method comprising the steps of: i) diagnosing T-cell lymphoma or leukemia in said subject using the method of any one of paragraphs 1-8; and ii) selecting a therapy based on the percentage of TRBC1- or TRBC2-positive T cells determined in step i), wherein said therapy is based on an isolated cell containing a chimeric antigen receptor (CAR) that contains an antigen-binding domain that selectively binds to TRBC1 or TRBC2. 10. A method for selecting a subject having a T-cell lymphoma or leukemia to receive therapy based on an isolated cell comprising a CAR that comprises an antigen-binding domain that selectively binds to TRBC1 or TRBC2, said method comprising the steps of: i) diagnosing T-cell lymphoma or leukemia in said subject using the method of any one of paragraphs 1-8; and ii) selecting said subject to receive said therapy based on the percentage of TRBC1- or TRBC2-positive T cells determined in step i). 11. The method according to any one of claims 9 or 10, characterized in that said sample is a peripheral blood sample or a biopsy.