ROR1 antibodies

Abstract

There is described Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) antibodies that specifically bind a ROR1 polypeptide, and their use. In particular, isolated monoclonal antibodies are described and their use in a number of applications, including in the detection, prevention and treatment of cancer.

Claims

1. An isolated monoclonal antibody comprising a light chain variable domain and a heavy chain variable domain wherein the light chain variable domain comprises a light chain complementarity determining region (LCDR)1, an LCDR2 and an LCDR3, and wherein the heavy chain variable domain comprises a heavy chain complementarity determining region (HCDR)1, an HCDR2 and an HCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 24; LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 26; and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 28; wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 115; HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 117; and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 252 (X.sub.1X.sub.2HRYNLFDS, wherein X.sub.1 is Ala or Thr and X.sub.2 is Ser, Lys or Arg), or wherein the LCDR1 comprises the amino acid sequence set forth as SEQ ID NO: 272, the LCDR2 comprises the amino acid sequence set forth as SEQ ID NO: 273, the LCDR3 comprises the amino acid sequence set forth as SEQ ID NO: 28, the HCDR1 comprises the amino acid sequence set forth as SEQ ID NO: 274, the HCDR2 comprises the amino acid sequence set forth as SEQ ID NO: 275, and the HCDR3 comprises the amino acid sequence set forth as SEQ ID NO: 276; and wherein the monoclonal antibody specifically binds to a ROR1 polypeptide.

2. An isolated monoclonal antibody according to claim 1, wherein when HCDR3 comprises an amino acid sequence having the sequence set forth as SEQ ID NO: 252 (X.sub.1X.sub.2HRYNLFDS, wherein X.sub.1 is Ala or Thr and X2 is Ser, Lys or Arg), HCDR3 comprises the amino acid sequence as set forth in SEQ ID NO: 119, 249, 250 or 251.

3. An isolated monoclonal antibody according to claim 1 wherein: (a) the light chain variable domain comprises a Light Chain Framework Region (LCFR)1 comprising the amino acid sequence as set forth as one of SEQ ID NO: 23, SEQ ID NO: 230, SEQ ID NO: 238 or SEQ ID NO: 239; an LCFR2 comprising the amino acid sequence as set forth as one of SEQ ID NO: 25, SEQ ID NO: 231, SEQ ID NO: 233 or SEQ ID NO: 236; an LCFR3 comprising the amino acid sequence as set forth as one of SEQ ID NO: 27, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 237 or SEQ ID NO: 240; and an LCFR4 comprising the amino acid sequence as set forth as one of SEQ ID NO: 14, SEQ ID NO: 205 or SEQ ID NO: 234, wherein the Light Chain Framework Regions may include up to 10 amino acid substitutions in the amino acid sequences as set forth above, or (b) the heavy chain variable domain comprises a Heavy Chain Framework Region (HCFR)1 comprising the amino acid sequence as set forth as one of SEQ ID NO: 114, SEQ ID NO: 215, SEQ ID NO: 218 or SEQ ID NO: 223; an HCFR2 comprising the amino acid sequence as set forth as one of SEQ ID NO: 116, SEQ ID NO: 216, SEQ ID NO: 219 or SEQ ID NO: 221; an HCFR3 comprising the amino acid sequence as set forth as one of SEQ ID NO: 118, SEQ ID NO: 217, SEQ ID NO: 220, SEQ ID NO: 222 or SEQ ID NO: 224; and an HCFR4 comprising the amino acid sequence as set forth as one of SEQ ID NO: 94, SEQ ID NO: 186 or SEQ ID NO: 108, wherein the Heavy Chain Framework Regions may include up to 10 amino acid substitutions in the amino acid sequences as set forth above.

4. An isolated monoclonal antibody according to claim 1 wherein: (a) the light chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NO: 79, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244 or SEQ ID NO: 245, or an amino acid sequence having at least 90% identity to the specified amino acid sequences; or (b) the heavy chain variable domain comprises the amino acid sequence as set forth as one of SEQ ID NO: 172, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228 or SEQ ID NO: 229, or an amino acid sequence having at least 90% identity to the specified amino acid sequences.

5. An isolated monoclonal antibody comprising a light chain variable domain and a heavy chain variable domain wherein: (a) the light chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 79 and the heavy chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 172; (b) the light chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 241 and the heavy chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 225; (c) the light chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 242 and the heavy chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 226; (d) the light chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 243 and the heavy chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 227; (e) the light chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 244 and the heavy chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 228; or (f) the light chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 245 and the heavy chain variable domain comprises the amino acid sequence as set forth as SEQ ID NO: 229.

6. The isolated monoclonal antibody of claim 1 or 5, wherein: (a) the antibody is an IgG, IgM or IgA; (b) the antibody is a humanised antibody; (c) the antibody binds to an epitope of ROR1, wherein the epitope comprises amino acid Gln-261; (d) the antibody is labeled; or (e) the antibody is labeled, wherein the label is a fluorescent, an enzymatic, or a radioactive label.

7. An isolated antigen binding fragment of the isolated monoclonal antibody of claim 1 or 5.

8. The isolated antigen binding fragment of claim 7, wherein: (a) the fragment is a Fab fragment, a Fab′ fragment, a F(ab)′2 fragment, a single chain Fv protein (scFv), or a disulfide stabilized Fv protein (dsFv); (b) the antigen binding fragment is a Fab or an scFv fragment; (c) the antigen binding fragment is labeled; (d) the antigen binding fragment is labeled, wherein the label is a fluorescent, an enzymatic, or a radioactive label; or (e) the antigen binding fragment is included in a bispecific antibody.

9. A composition comprising the antibody of claim 1 or 5, or an antigen binding fragment thereof, and a pharmaceutically acceptable carrier.

10. An isolated nucleic acid encoding the monoclonal antibody of claim 1 or 5, or an antigen binding fragment thereof.

11. An isolated host cell transformed with a nucleic acid encoding the monoclonal antibody of claim 1 or 5, or an antigen binding fragment thereof.

12. A method of detecting cancer in a subject comprising: contacting a biological sample from the subject with at least one isolated monoclonal antibody of claim 1 or 5, or an antigen binding fragment thereof; and detecting antibody bound to the sample, wherein the presence of antibody bound to the sample indicates that the subject has cancer.

13. The method of detecting of claim 12, wherein: (a) the antibody specifically binds a ROR1 polypeptide, and wherein the presence of antibody bound to the sample indicates that the subject has leukemia, pancreatic cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, glioblastoma, testicular cancer, uterine cancer, adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma, sarcoma or renal cancer; (b) the isolated monoclonal antibody is directly labeled; or (c) the method further comprises: contacting the sample with a second antibody that specifically binds the isolated monoclonal antibody; and detecting the binding of the second antibody, wherein an increase in binding of the second antibody to the sample as compared to binding of the second antibody to a control sample detects the presence of cancer in the subject.

14. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one antibody of claim 1 or 5, or an antigen binding fragment thereof, thereby treating cancer.

15. The method of treating cancer of claim 14, wherein the cancer is leukemia, pancreatic cancer, prostate cancer, colon cancer, bladder cancer, ovarian cancer, glioblastoma, testicular cancer, uterine cancer, adrenal cancer, breast cancer, lung cancer, melanoma, neuroblastoma, sarcoma or renal cancer.

16. A kit comprising an antibody according to claim 1 or 5, or an antigen binding fragment thereof.

17. A kit according to claim 16 wherein: (a) the antibody or fragment is directly labelled; or (b) the kit further comprises an immunoassay.

18. A method of detecting cancer of claim 13, wherein the leukemia is Chronic Lymphocytic Leukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), Mantle Cell Leukaemia or Hairy Cell Leukaemia.

19. A method of treating cancer of claim 15, wherein the leukemia is Chronic Lymphocytic Leukaemia (CLL), Acute Lymphoblastic Leukaemia (ALL), Mantle Cell Leukaemia or Hairy Cell Leukaemia.

Description

(1) The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described. The examples should be read in combination with the figures which are as follows:

(2) FIG. 1: Identification of ROR1 binding domain: (A) SUP-T1 cells were transduced with retroviral vectors containing either the full extracellular portion of ROR1 or only one or two extracellular domains. Non-transduced SUP-T1 (SUP-T1 NT) cells served as negative control. (B) All 12 chimeric antibodies and (C) all 10 positive clones in scFv format were incubated with SUP-T1 NT and the new stable cell lines at 4° C. for 30 min. Cells were washed and stained with a secondary antibody (anti-human Fc-Dylight647), which was used as negative control. eROR1=extracellular ROR1, Ig-like=Immunoglobulin-like domain, Fz=Frizzled domain, Kg=Kringle domain.

(3) FIG. 2: Humanization of rat scFvs: A total of 25 constructs per clone were generated by combining five VH and five VL. All 25 scFv constructs were tested on ROR1.sup.+ and ROR1.sup.− cell lines. Secondary antibody alone and media alone served as negative controls, whilst the parental versions of each clone acted as positive control.

(4) FIG. 3: K.sub.D determination by surface plasmon resonance. Sensorgrams obtained using a Biacore X100 instrument are shown. Briefly, ROR1 antibodies were immobilized using a CMS chip and seven different concentrations of the Histidine-tagged ROR1 protein (extracellular region) were injected. Concentrations ranged from 1.5 to 100 nm.

(5) FIG. 4: Complement-dependent cytotoxicity of ROR1 MAbs on CLL cells (n=3). (A) Chimeric ROR1 MAbs supernatants were tested at 0.5 ug/ml on CLL cells and PBMCs. Only clone A showed a significant toxicity compared to the isotype (p<0.001). Rituximab (Rtx) was used as positive control at 0.5 ug/ml and 10 ug/ml, and achieved significant cytotoxicity on CLL1 and CLL2 samples (p<0.001). Cytotoxicity on CLL samples was normalized to PBMCs' data. (B) Cell surface staining for CD20 and ROR1 by flow cytometry. The red area represents the isotype control. Error bars in (A) represent SD. Experiments were done in triplicates.

(6) FIG. 5: Epitope mapping of ROR1 clone A MAb. (A) Reactivity of clone A with ROR1-derived overlapping peptides was analyzed by ELISA. (B) Amino acid substitution within the epitope binding region was performed in ROR1-transduced cell lines. Also, the previously described clones R12, 4a5 and D10 were also included for comparison. Red circles indicate essential amino acids for antibody binding. One out of three experiments is shown. Experiments were done in triplicates. Error bars represent SD.

(7) FIG. 6: Epitope mapping of ROR1 clone F MAb. (A) Reactivity of clone F with ROR1-derived overlapping peptides was analyzed by ELISA. Since no linear epitope was identified, (B) amino acid substitution of non-conserved regions within the Fz domain was performed in ROR1-Fz transduced cell lines. Circles indicate essential amino acids for antibody binding. One out of three experiments is shown. Experiments were done in triplicates. Error bars represent SD.

(8) FIG. 7: Competition assay by flow cytometry. Clone A (SA1) in murine IgG2a, k (mSA1) along with other ROR1 MAbs in human IgG1, k—including previously published 4a5, D10 and R12 clones—were used for staining ROR1-transduced cells. MAb staining was done as (A) single agents or (B) in combination with clone A (mSA1). Anti-human IgG (450) and anti-mouse IgG (DyLight649) were used as secondary antibodies. Combined staining using clones F and mSA1 acted as negative control for overlapping epitopes.

(9) FIG. 8: Internalisation of ROR1 antibodies on SKW 6.4 GFP cells by flow cytometry. SKW 6.4 GFP cells were stained at 4° C. and either kept on ice (blue line) or incubated at 37° C. (yellow line) for 1 h. Of all tested MAbs, clone V showed a pronounced reduction in MFI (green circle). Clones A, F and Mu (purple circle) were selected for further investigation. Clones R12 and 4a5 (in black) served as negative and positive control, respectively. Anti-human Fc-Dylight 647 was used as secondary antibody and as staining control (red line). MFI=Median Fluorescence Intensity.

(10) FIG. 9: Internalisation of ROR1 MAbs on SKW 6.4 GFP and CLL cells by flow cytometry. Cells were stained at 4° C. and either left on ice or incubated at 37° C. for 15 min, 1 h and 2 h. Cells were then analysed using an anti-human IgG as secondary antibody. (A) Histograms and (B) Trends over time showing the MFI reduction over time are presented. The % of MFI reduction was calculated as described in the Materials and Methods chapter. Phenylarsine oxide (PAO), an endocytosis inhibitor, acted as negative control (PAO-120).

EXAMPLES

Example 1

(11) Immunization of Rats and Production of ROR1 Antibodies

(12) A total of 3 Wistar rats were immunized using a DNA-based protocol. Briefly, human ROR1 coding sequence cloned into an immunization plasmid was introduced into the rats, the target protein was expressed and an immune response was generated. Four applications of DNA using a gene gun were initially performed. Rat serum was then analyzed, followed by 4 additional applications. After confirming that serum from all three challenged rats showed presence of anti-human ROR1 antibodies by flow cytometry, animals were sacrificed after 102 days of immunization. Lymph nodes were removed and pooled in order to produce oligoclonal hybridoma cell lines.

(13) A total of 38 positive hybridomas were identified by testing their binding ability to cells transfected with either pB1-ROR1-hum or with an irrelevant construct. As before, this was assessed by flow cytometry.

(14) Antibody sequences were obtained by 5′ Rapid Amplification of cDNA ends. Oligoclonal hybridomas from Aldevron GmBH were separated into single cell clones either by dilution or single cell sorting into 96 well plates and colonies grown until confluent (approximately 2 weeks). Supernatant was screened against ROR1 positive and negative cell lines to ensure the presence of a specific anti-ROR1 antibody and also used for isotyping using rat immunoglobulin isotyping kits (eBioscience or BD Bioscience).

(15) Clones were grown until confluence in 6 well plates or 10 cm plates and then pelleted into RNAlater (Life Technologies) before RNA was extracted using RNA MiniPlus Kit (Qiagen). RNA was reverse transcribed to cDNA using Quantitect Reverse Transcriptase (Qiagen). An aliquot of this cDNA was assessed with GAPDH primers which were able to differentiate genomic and cDNA to ensure quality of samples. cDNA had a polyC tail added with Terminal Transferase (New England Biolabs) and nested PCR reactions were performed (Phusion Taq; New England Biolabs or Platinum Taq High Fidelity: Life Technologies) to identify the variable regions of the heavy and light chains, using primers specific for light chain isotype and heavy chain isotype.

(16) PCR products were run on a 1% TBE gel and post-stained with Gelstar (Lonza). Bands of the correct size were extracted and sent for direct sequencing or inserted into Topo subcloning vectors (Life Technologies) for subsequent sequencing.

(17) Sequence data was compared to the IMGT V-QUEST database of Rat germline immunoglobulin sequences and consensus sequences obtained that were productive and had an in frame signal sequence (Brochet et al., 2008, Alamyar et al., 2012). Overlap extension primers were designed to amplify the heavy and light chains whilst introducing a linker sequence to generate ScFv constructs. A secreted version of the ScFv was produced by cloning the ScFv sequence in frame with murine IgG2a constant region using NcoI and BamHI sites (or if needed the compatible Bg1II or B1I sites).

(18) Antibodies were generated by cloning the variable sequence in frame with human IgG1 or mouse heavy IgG2a chain constant regions, and light chain with the corresponding human or mouse kappa constant regions. NcoI and MluI sites (or if needed, the compatible BspHI or PciI sites) were used. For humanized antibodies, variable regions were cloned in frame with human heavy IgG1 and light kappa constant regions.

(19) A total of 17 novel scFvs were generated. Out of these, 12 clones bound as antibodies and 10 bound in a single chain variable fragment (scFv) format (See Table 1). Additionally, to identify the binding domain of all positive clones, stable cell lines expressing either the full extracellular region of ROR1 (eROR1) or varying regions of its extracellular domains: Ig-like alone, Ig-like+Frizzled (Fz), Fz alone, Fz+Kringle (Kg) and Kg alone were generated by transducing SUP-T1 cells with retroviral supernatants (See FIG. 1A).

(20) In FIGS. 1B and 1C, we present flow cytometry data showing the binding domains of all 12 antibodies and 10 scFvs, respectively.

(21) TABLE-US-00001 TABLE 1 Binding Binds as whole Binds in Domain immunoglobulin scFv format Clone G3 Ig Yes No Clone G5 Ig Yes Yes Clone E7 Ig Yes No Clone J Ig Yes No Clone F Fz Yes Yes Clone B Ig Yes Yes Clone A Ig Yes Yes Clone I Ig Yes Yes Clone O Ig Yes Yes Clone Pi Ig Yes Yes Clone Mu Ig Yes Yes Clone R Ig Not tested Yes Clone V Between Yes Yes Ig and Fz

(22) As can be seen in FIG. 1C, in addition to the 10 scFv clones, two known ROR1 antibodies (R12 and 4A5) were also tested.

(23) With the exception of clone F, which binds the frizzled domain and clone V, which binds only the Ig-Fz protein, all other clones bound the immunoglobulin domain. Interestingly, and somewhat surprisingly, the prior art antibodies R12 and 4A5 show different and distinct binding characteristics to Clone F.

Example 2

(24) Humanisation of Rat scFvs

(25) Clones A and F were selected for humanisation. The variable domain sequences of rat scFvs were searched against the human IgG germline database. Five human framework sequences with high homology to each rat antibody were chosen as human acceptors for both light and heavy chains CDRs. The sequences of five humanized VLs and humanized VHs were obtained after directly grafting the CDRs of each rat antibody to the human acceptor frameworks.

(26) For each clone, a total of 25 constructs were generated by combining all five VH and five VL. Cloning was performed as described above. Binding was then tested on ROR1.sup.+ and ROR1.sup.− cell lines. Parental versions of clone A and F scFvs served as positive control for antibody staining, whilst media alone and secondary antibody alone acted as negative controls (See FIG. 2).

Example 3

(27) Binding Efficiencies

(28) We undertook surface plasmon resonance evaluation using a Biacore X100 instrument. ROR1 chimeric antibodies were immobilised using an anti-human IgG1 antibody capture kit and a CMS sensor chip. Seven different concentrations, ranging from 1.5-100 nM, of the extracellular portion of ROR1 bearing a Histidine tag were then injected.

(29) Surface plasmon resonance analysis showed that clones B5, A and Pi possessed the strongest affinities (1.51, 1.81 and 1.98 nM, respectively), whilst clone F presented the weakest one (5.46 nM).

(30) Table 2 below further illustrates the binding kinetics of our ROR1 chimeric antibodies.

(31) Affinity Values (K.sub.D) of Chimeric ROR1 Antibodies

(32) TABLE-US-00002 ROR1 K.sub.on, K.sub.off, K.sub.D, Antibody (10.sup.5)M.sup.−1s.sup.−1 (10.sup.−4)s.sup.−1 10.sup.−9M E7 4.168 8.490 2.037 G3 1.086 2.660 2.450 G5 1.593 4.164 2.614 A 5.774 10.44 1.809 B5 4.639 6.980 1.505 F 1.531 8.362 5.460 J6 2.492 8.351 3.351 I 2.093 5.100 2.436 Pi 1.763 3.487 1.979 O 1.735 6.075 3.501

Example 4

(33) Anti-Human ROR1 Antibody Cytotoxicity on Chronic Lymphoid Leukemia (CLL) Cells-Complement-Dependent Cytotoxicity (CDC) Assay

(34) A CDC assay of the antibodies described above was carried out. Two antibodies from other groups, namely R12 and 4A5, were also tested to see how the antibodies of the invention compared to previously published antibodies. Antibodies were generated with human IgG1 Fc domain. The antibodies were incubated for two hours with CLL cells, after which cytotoxicity on the cells was determined.

(35) Antibodies were tested at a concentration of 0.5 ug/ml. The isotype and Rituximab (Rtx) controls were therefore used at this concentration. For Rituximab however we used an additional concentration of 10 ug/ml in order to have a true positive control for the assay.

(36) In FIG. 4A, CDC activity elicited by all 12 of our own ROR1 chimeric antibodies can be observed. Of all antibodies evaluated, clone A was the only one that showed significant cytotoxicity compared to the isotype (p<0.001). Remarkably, clone A was also better at killing CLL cells than the known ROR1 antibodies R12 and 4a5 but was still not as CDC active as Rituximab.

(37) The expression levels of both CD20 and ROR1 antigens on the surface of CLL samples was also assessed. FIG. 4B explains why Rituximab is able to kill CD20 expressing cells very efficiently (CLL1 and 2), but not CLL3, as the latter was virtually negative for CD20 expression.

Example 5

(38) Epitope Mapping

(39) The epitopes of clones A and F described above were mapped by two approaches: peptide library ELISA and amino acid substitution.

(40) Binding of clones A and F on overlapping peptides, covering all three extracellular domains of ROR1, was tested by ELISA. This approach allowed us to narrow down the epitope of clone A to a five amino acid region within the Ig-like domain (FIG. 5A).

(41) Point mutations were then generated for the Ig-like domain of human ROR1 at positions 47 to 51. The particular mutations used were N(47)F, I(48)Q, S(50)D and the substitution of the five amino acid sequence NISSE (SEQ ID NO: 253) at residues 47 to 51 with the sequence FQDDL (SEQ ID NO: 254).

(42) Retroviral vectors were used for transducing SUP-T1 cells and transduction was tested by GFP expression using flow cytometry.

(43) It was found that the N(47)F and the I(48)Q substitutions reduced or stopped the clone A antibody binding to ROR1-Ig like domain, whereas the S(50)D substitution did not seem to affect binding. Further, the five amino acid substitution also prevented antibody binding. Therefore, it seems that Asn-47 and Ile-48 are essential for antibody binding. It was found that this epitope is unique to clone A and there is no overlap with previously published ROR1 clones (R12, 4a5 and D10, the prototype of Cirmtuzumab). Results can be seen in FIG. 5B.

(44) For clone F, peptide library ELISA data showed that it was not possible to detect any binding signal above background (FIG. 6A), suggesting that this antibody does not bind to a linear sequence, rather it might potentially recognize a conformational epitope. Point mutations were therefore generated for the Fz domain of human ROR1 at non-conserved regions (positions 254 and 261). The particular mutations used were I(254)V and Q(261)H.

(45) Retroviral vectors were used for transducing SUP-T1 cells and transduction was tested by GFP expression using flow cytometry.

(46) It was found that the Q(261)H substitution reduced or stopped the clone F antibody binding to ROR1-Fz domain, whereas the I(254)V substitution did not seem to affect binding. Further, the combination of Q(261)H and I(254)V also prevented antibody binding. Therefore, it seems that Gln-261 is essential for antibody binding. Results can be seen in FIG. 6B.

Example 6

(47) Competition Assay by Flow Cytometry

(48) To further challenge our previous observations—whereby clone A had a distinct epitope that was not shared with other anti-ROR1 antibodies—we decided to compare the binding of A to ROR1 with other relevant clones through a flow cytometry-based competition assay.

(49) Based on previous studies, we hypothesised that consecutive- and/or simultaneous-staining of ROR1.sup.+ cells, using clone A in combination with other anti-ROR1 antibodies, would allow us to identify overlapping epitopes by flow cytometry analysis.

(50) To this end, we fused the variable region of clone A to a mouse IgG2a, kappa constants following the same protocols discussed in previous chapters. All other chimeric antibodies were kept in their existing rat-human format. We needed to generate A in a distinct constant region in order to allow simultaneous and specific detection of every pair of clones we were testing. In other words, since we were investigating unlabelled clones, a secondary antibody staining against clones possessing different isotypes was required. Also, for ease of analysis, ROR1.sup.+ GFP.sup.+ cells were used.

(51) FIG. 7A shows strong ROR1 binding when antibodies were used as single agents, which confirmed their correct expression and detection of ROR1. Since A and F antibodies detect different extracellular domains, this combination was included in our studies as negative control for overlapping epitopes.

(52) In FIG. 7B, three different columns are presented. Dot plots on the first column correspond to ROR1 binding assessed when SUP-T1 ROR1 cells were stained with clone A (SA1) (mIgG) in the first instance. After a washing step, cells were stained with a competitor antibody (hIgG), followed by another washing step. Both antibodies were then detected by a third staining step using the appropriate secondary antibodies. In the second column, a similar approach was taken when staining these cells, except that this time it was the antibody in hIgG1, k format the one used for the first staining step, followed by clone A (SA1) staining. On the third column, both antibodies were used at the same time.

(53) In all cases, a clear and defined shift of all events was observed; indicating that, independent of the order in which cells were stained, antibodies used in the first staining step did not impede binding of the antibodies used in the second step. This was further confirmed when both antibodies were assessed at the same time. Hence, these results confirm that clone A binds to a unique epitope within the Ig-like domain, which is not shared (not even partially) with other reported clones.

Example 7

(54) Assessment of Internalisation

(55) Antibody internalisation can have important implications as it could provide us with the opportunity to develop armed antibodies that could be conjugated to toxic payloads, such as toxins, radioisotopes or chemotherapeutic agents. This class of therapeutic antibodies are called Antibody-Drug Conjugates (ADCs). Interestingly, ADCs could have a therapeutic advantage over naked antibodies (unconjugated), in terms of potency and efficacy, as their cytotoxicity relies on the payload they carry rather than the immune system of patients. Thus, we next investigated whether our antibodies were able to get internalised into ROR1.sup.+ cells by flow cytometry and pH-Amine dye labelling.

(56) By Flow Cytometry

(57) SKW 6.4 cells, Epstein-Barr virus-transformed B cells endogenously expressing comparable levels of ROR1 as CLL patients, were incubated on ice with all 12 ROR1 chimeric antibodies. After 30 min, cells were washed with ice-cold PBS, and either left on ice or incubated at 37° C. for 1 h. Subsequent staining with an anti-human Fc-Dylight 647 was used to detect any primary antibody that had remained on the cell surface. Previously reported clones R12 and 4a5 were also included in this assay as negative and positive controls, respectively (FIG. 8).

(58) From all tested antibodies, clone V (green circle) showed almost complete MFI reduction. A modest but detectable decrease in MFI levels was observed in clones A and F (purple circles), similar to the one detected for clone 4a5. As previously reported, clone R12 did not show significant MFI reduction after 1 h incubation at 37° C.

(59) MFI reduction could be caused by dissociation or internalisation or a combination of both (15). In order to further investigate what was triggering this drop in MFI for clones V, A and F, we decided to use an endocytosis inhibitor in our next experiments.

(60) Endocytosis Inhibition

(61) Phenylarsine oxide (PAO), a trivalent arsenical compound, is the typical chemical chosen to block Clathryn-mediated endocytosis (CME), although it can also inhibit macropinocytosis and phagocytosis. CME is the best studied mechanism of endocytosis, and it has been established that receptor tyrosine kinases (RTKs), such as ROR1, predominantly use this form of internalisation when engulfed by the cell membrane and drawn inside the cell.

(62) To distinguish between dissociation and internalisation, SKW 6.4 cells were incubated on ice in the presence of our selected antibodies for 30 min. Cells were then washed with ice-cold PBS and either left on ice or incubated at 37° C. for 15 min, 1 h or 2 h. For the 2 h time point, a duplicate sample was incubated with PAO (10 μM). Immediately after incubation, all samples were washed with ice-cold PBS and stained with an anti-human Fc-Dylight 647 for 30 min. Cells were analysed by flow cytometry and MFI reduction was calculated.

(63) Flow cytometry analysis showed that although clone V had an important MFI reduction, it was mainly due to dissociation as PAO did not considerably block the drop in MFI after 2 h incubation at 37° C. A combination of internalisation and dissociation was more evident for the other 3 clones, being internalisation the dominating factor for clone 4a5. This was even more evident for clone A.

(64) To verify these results, we repeated the experiment using SKW 6.4 cells and 2 samples of primary cells from CLL patients, expressing either high or low levels of ROR1 (FIG. 9). Samples were processed as mentioned above and analysed by flow cytometry. Interestingly, these data confirmed that whilst both dissociation and internalisation were involved in the MFI reduction, for clone V dissociation was the main reason for decrease in MFI. This was even clearer on CLL cells, where virtually no internalisation of clone V was detected.

(65) The dissociation of clones F, 4a5 and A from the cell surface of CLL cells was very similar between samples and seemed to be independent of ROR1 levels. In this case, internalisation appeared to be the main contributing factor to MFI reduction. A similar observation was detected on SKW 6.4 cells; although on this cell model, clone A was the only antibody where MFI drop was almost completely blocked by PAO and only partially for clones F and 4a5, suggesting that although these last 3 antibodies might get partially internalised, clone A might be the most promising one.

Example 8

(66) Clone F is Unique to Other Antibodies Generated (Murine and Rabbit) Because of Sequence Homology

(67) Human, murine, rabbit and rat ROR1 protein sequences were aligned using Uniprot web based software (http://www.uniprot.org/align/) and the variation between the different species highlighted. Uniprot accession numbers: Human (Q01973), Murine (Q9Z139) and Rabbit (G1U5L1). For rat ROR1, NCBI reference sequence NP 001102141.1 was used as the corresponding Uniprot sequence was only partially complete.

(68) Clone F binds to Q261, which was possible due to differences between rat and human amino acids at this position (the human amino acid at position 261 is glutamine (Q) whereas the corresponding amino acid at this position in rat is histidine (H)). When rats are immunised with human ROR1, this amino acid difference is recognised as an immunogen relative to the rat ROR1 sequence, against which an antibody is produced.

(69) The known antibody R12 (rabbit) and murine ROR1 binders show homology with human ROR1 at this site (i.e. they all have glutamine (Q) at this position). As a result, immunisation of rabbits or mice with human ROR1 does not result in antibody production directed to this position as it is not immunogenic. In view of this, clone F is unique in its ability to bind to this epitope.

Example 9

(70) Humanisation Imparts Advantages Compared to Non-Humanised Comparator Constructs

(71) One of the rationales for targeting ROR1 as opposed to CD19, is sparing of the normal ROR1 negative B cell population. However at the same time, continued presence of normal CD19+ B cells allows for immune responses directed against a rat derived scFv. This has been seen with murine scFvs and have led to clinically significant outcomes, including anaphylaxis with mRNA modified mesothelin CAR T cells (Maus et al., 2013) or antibody responses, with α-folate receptor or carbonic anhydrase IX specific CAR T cells (Lamers et al., 2006, Kershaw et al., 2006). T cell mediated immune responses are also possible due to cross presentation of components of the CAR on MHC. CD19 CART cells by comparison, inherently diminish the risk of antibody based immune responses by eradicating the normal B cell population, with B cell recurrence associated with a higher risk of relapse. By undertaking humanisation, we have decreased the likelihood of immune responses against the antibody leading to enhanced persistence and decreased immunogenicity.

(72) TABLE-US-00003 Sequence Listing The amino acid sequences listed are shown in the application using standard one  letter codes for amino acids. The sequences below relate to 13 clones (G3, G5, E7, J, F, B, A, I, O, Pi, Mu, R and V) that were developed. Also  contained in the table below are sequences relating to the humanisation of  clones A and F. SEQ ID Sequence Clone Description NO: DVVMTQTPVSLPVSLGGQVSISCRSS G3 Rat Light Chain Framework Region 1 1 QSLEHSNGDTF G3 Light Chain CDR1 2 LHWYLQKPGQSPRLLIY G3 Rat Light Chain Framework Region 2 3 RVS G3 Light Chain CDR2 4 NRFSGVPDRFSGSGSGTDFTLKISRIEPEDLG G3 Rat Light Chain Framework Region 3 5 DYYC LQSTHFPNT G3 Light Chain CDR3 6 FGAGTKLELK G3 Rat Light Chain Framework Region 4 7 DIQLTQSPSTLSASLGERVTISCRAS G5 Rat Light Chain Framework Region 1 8 QSISNS G5 Light Chain CDR1 9 LNWYQQKPDGTVKRLIY G5 Rat Light Chain Framework Region 2 10 STS G5 Light Chain CDR2 11 TLESGVPSRFSGSGSGTDFSLSISSLESEDFAM G5 Rat Light Chain Framework Region 3 12 YYC LQFATYPQVT G5 Light Chain CDR3 13 FGSGTKLEIK G5 Rat Light Chain Framework Region 4 14 DIVLTQSPALAVSVGQRATISCRAS E7 Rat Light Chain Framework Region 1 15 QSVSISRYNF E7 Light Chain CDR1 16 MHWYQQKPGQQPKLLIY E7 Rat Light Chain Framework Region 2 17 RAS E7 Light Chain CDR2 18 NLASGIPARFSGSGSGTDFTLTINPVQADDIA  E7 Rat Light Chain Framework Region 3 19 TYYC QQNRESPRT E7 Light Chain CDR3 20 FGGGTKLELK E7 Rat Light Chain Framework Region 4 21 DIVLTQSPALAVSVGQRATISCRAS J Rat Light Chain Framework Region 1 15 QSVSISRYDF J Light Chain CDR1 22 MHWYQQKPGQQPKLLIY J Rat Light Chain Framework Region 2 17 RAS J Light Chain CDR2 18 NLASGIPARFSGSGSGTDFTLTINPVQADDIA  J Rat Light Chain Framework Region 3 19 TYYC QQNRESPRT J Light Chain CDR3 20 FGGGTKLELK J Rat Light Chain Framework Region 4 21 DIQMTQSPSFLSASVGDRVTINCKAS F Rat Light Chain Framework Region 1 23 QNIDRY F Light Chain CDR1 24 LNWYQQKLGEAPKRLLY F Rat Light Chain Framework Region 2 25 NTN F Light Chain CDR2 26 KLQTGIPSRFSGSGSATDFTLTISSLQPEDFAT F Rat Light Chain Framework Region 3 27 YFC LQYNSLPLT F Light Chain CDR3 28 FGSGTKLEIK F Rat Light Chain Framework Region 4 14 DIQMTQSPSSMSASLGDRVTFTCQAS A Rat Light Chain Framework Region 1 29 QDIGNN A Light Chain CDR1 30 LIWFQQKPGKSPRPLMY A Rat Light Chain Framework Region 2 31 FAT A Light Chain CDR2 32 SLANGVPSRFSGSRSGSDYSLTISSLESEDLAD A Rat Light Chain Framework Region 3 33 YHC LQYREYPLT A Light Chain CDR3 34 FGSGTKLDLK A Rat Light Chain Framework Region 4 35 DIRMTQSPASLSASLGETVTIECLTS B Rat Light Chain Framework Region 1 36 EDIYSD B Light Chain CDR1 37 LAWFQQKPGKSPQLLIY B Rat Light Chain Framework Region 2 38 DAN B Light Chain CDR2 39 SLQNGVPSRFGGCGSGTQYSLQISSLQSEDV B Rat Light Chain Framework Region 3 40 ATYFC QQYKNYPPT B Light Chain CDR3 41 FGGGTKLVLK B Rat Light Chain Framework Region 4 42 DIQLTQSPSSMSASLGDRVSLTCQSS I Rat Light Chain Framework Region 1 43 QGIGKY I Light Chain CDR1 44 LSWYQHKPGKPPKAMIY I Rat Light Chain Framework Region 2 45 YAT I Light Chain CDR2 46 KLADGVPSRFSGSRSGSDFSLTISSLESEDIAIY I Rat Light Chain Framework Region 3 47 YC LQFDDYPWT I Light Chain CDR3 48 FGGGTKLELK I Rat Light Chain Framework Region 4 21 DIVLTQSPALAVSLEQRVTIACKTS O Rat Light Chain Framework Region 1 49 QNVDNHGISY O Light Chain CDR1 50 MHWYQQKSGQEPKLLIY O Rat Light Chain Framework Region 2 51 EGS O Light Chain CDR2 52 NLAVGIPARFSGSGSGTDFTLTIDPVEADDIE O Rat Light Chain Framework Region 3 53 TYYC QQSKDDPRT O Light Chain CDR3 54 FGGGTKLELK O Rat Light Chain Framework Region 4 21 QFTLTQPKSVSGSLRSTITIPCERS R Rat Light Chain Framework Region 1 55 SGDIGDSY R Light Chain CDR1 56 VSWYQQHLGRPPINVIY R Rat Light Chain Framework Region 2 57 ADD R Light Chain CDR2 58 QRPSEVSDRFSGSIDSSSNSASLTITNLQMDD R Rat Light Chain Framework Region 3 59 EADYFC QSYDRNVDFNTV R Light Chain CDR3 60 FGGGTKVTVL R Rat Light Chain Framework Region 4 61 DIQLTQSPSSLSASLGDRVSLTCQSS Pi Rat Light Chain Framework Region 1 62 QGIGKY Pi Light Chain CDR1 44 LSWFQHKPGKPPKPVIN Pi Rat Light Chain Framework Region 2 63 YAT Pi Light Chain CDR2 46 NLADGVPSRFSGRRSGSDFSLTISSLESEDTAI Pi Rat Light Chain Framework Region 3 64 YYC LQFDDFRWT Pi Light Chain CDR3 65 VGGGTKLELK Pi Rat Light Chain Framework Region 4 66 QFTLTQPKSVSGSLRSTITIPCERS Mu Rat Light Chain Framework Region 1 55 SGDIGDNY Mu Light Chain CDR1 67 VSWYQQHLGRPPINVIY Mu Rat Light Chain Framework Region 2 57 ADD Mu Light Chain CDR2 58 QRPSEVSDRFSGSIDSSSNSASLTITNLQMDD Mu Rat Light Chain Framework Region 3 59 EADYFC QSFDSNFDIPV Mu Light Chain CDR3 68 FGGGTKLTVL Mu Rat Light Chain Framework Region 4 69 DIKMTQSPSFLSASVGDRVTINCKAS V Rat Light Chain Framework Region 1 70 QNITRF V Light Chain CDR1 71 LNWYQQELGEAPTLLIY V Rat Light Chain Framework Region 2 72 NTN V Light Chain CDR2 26 NLQTGIPSRFSGSGSGTDFTLTISSLQPEDVA V Rat Light Chain Framework Region 3 73 TYFC LQHGSRPRT V Light Chain CDR3 74 FGGGTKLELK V Rat Light Chain Framework Region 4 21 DVVMTQTPVSLPVSLGGQVSISCRSSQSLEH G3 Rat Light Chain Variable Region 75 SNGDTFLHWYLQKPGQSPRLLIYRVSNRFSG VPDRFSGSGSGTDFTLKISRIEPEDLGDYYCL QSTHFPNTFGAGTKLELK DIQLTQSPSTLSASLGERVTISCRASQSISNSL G5 Rat Light Chain Variable Region 76 NWYQQKPDGTVKRLIYSTSTLESGVPSRFSG SGSGTDFSLSISSLESEDFAMYYCLQFATYPQ VTFGSGTKLEIK DIVLTQSPALAVSVGQRATISCRASQSVSISRY E7 Rat Light Chain Variable Region 77 NFMHWYQQKPGQQPKLLIYRASNLASGIPA RFSGSGSGTDFTLTINPVQADDIATYYCQQN RESPRTFGGGTKLELK DIVLTQSPALAVSVGQRATISCRASQSVSISRY J Rat Light Chain Variable Region 78 DFMHWYQQKPGQQPKLLIYRASNLASGIPA RFSGSGSGTDFTLTINPVQADDIATYYCQQN RESPRTFGGGTKLELK DIQMTQSPSFLSASVGDRVTINCKASQNIDR F Rat Light Chain Variable Region 79 YLNWYQQKLGEAPKRLLYNTNKLQTGIPSRF SGSGSATDFTLTISSLQPEDFATYFCLQYNSLP LTFGSGTKLEIK DIQMTQSPSSMSASLGDRVTFTCQASQDIG A Rat Light Chain Variable Region 80 NNLIWFQQKPGKSPRPLMYFATSLANGVPS RFSGSRSGSDYSLTISSLESEDLADYHCLQYRE YPLTFGSGTKLDLK DIRMTQSPASLSASLGETVTIECLTSEDIYSDL B Rat Light Chain Variable Region 81 AWFQQKPGKSPQLLIYDANSLQNGVPSRFG GCGSGTQYSLQISSLQSEDVATYFCQQYKNY PPTFGGGTKLVLK DIQLTQSPSSMSASLGDRVSLTCQSSQGIGK I Rat Light Chain Variable Region 82 YLSWYQHKPGKPPKAMIYYATKLADGVPSRF SGSRSGSDFSLTISSLESEDIAIYYCLQFDDYP WTFGGGTKLELK DIVLTQSPALAVSLEQRVTIACKTSQNVDNH O Rat Light Chain Variable Region 83 GISYMHWYQQKSGQEPKLLIYEGSNLAVGIP ARFSGSGSGTDFTLTIDPVEADDIETYYCQQS KDDPRTFGGGTKLELK QFTLTQPKSVSGSLRSTITIPCERSSGDIGDSY R Rat Light Chain Variable Region 84 VSWYQQHLGRPPINVIYADDQRPSEVSDRFS GSIDSSSNSASLTITNLQMDDEADYFCQSYD RNVDFNTVFGGGTKVTVL DIQLTQSPSSLSASLGDRVSLTCQSSQGIGKYL Pi Rat Light Chain Variable Region 85 SWFQHKPGKPPKPVINYATNLADGVPSRFS GRRSGSDFSLTISSLESEDTAIYYCLQFDDFR WTVGGGTKLELK QFTLTQPKSVSGSLRSTITIPCERSSGDIGDNY Mu Rat Light Chain Variable Region 86 VSWYQQHLGRPPINVIYADDQRPSEVSDRFS GSIDSSSNSASLTITNLQMDDEADYFCQSFDS NFDIPVFGGGTKLTVL DIKMTQSPSFLSASVGDRVTINCKASQNITRF V Rat Light Chain Variable Region 87 LNWYQQELGEAPTLLIYNTNNLQTGIPSRFS GSGSGTDFTLTISSLQPEDVATYFCLQHGSRP RTFGGGTKLELK EVQLQESGPGLVKPAQSLSLTCSVT G3 Rat Heavy Chain Framework Region 1 88 GYSITNMYR G3 Heavy Chain CDR1 89 WNWIRKFPGNKLEWMGY G3 Rat Heavy Chain Framework Region 2 90 INTAGST G3 Heavy Chain CDR2 91 DYSPSLRGRVSITGDTSKNQFFLHLTSVTTED G3 Rat Heavy Chain Framework Region 3 92 TATYYC AGFITNPFDF G3 Heavy Chain CDR3 93 WGQGVMVTVSS G3 Rat Heavy Chain Framework Region 4 94 EVQVVESGGGLVQPGRSLKLSCVPS G5 Rat Heavy Chain Framework Region 1 95 GFTFNNYW G5 Heavy Chain CDR1 96 MTWIRQAPGKAPEWVAS G5 Rat Heavy Chain Framework Region 2 97 ISNTGGST G5 Heavy Chain CDR2 98 FYPDSVRGRFSISRDNTKGTLYLHMTSLRSED G5 Rat Heavy Chain Framework Region 3 99 TATYYC IRNMDA G5 Heavy Chain CDR3 100 WGQGTSVTVSS G5 Rat Heavy Chain Framework Region 4 101 GKLVESGGGLLKPGGSLKLSCVAS E7 Rat Heavy Chain Framework Region 1 102 GFTFDKYW E7 Heavy Chain CDR1 103 MHWVRQAPGKGLEWIAE E7 Rat Heavy Chain Framework Region 2 104 IEYDGTET E7 Heavy Chain CDR2 105 NYAPSIKDRFTISRDNAKNTLYLQMSNVRSE E7 Rat Heavy Chain Framework Region 3 106 DAATYFC TTEEMYTTDYYYGFAY E7 Heavy Chain CDR3 107 WGQGTLVTVSS E7 Rat Heavy Chain Framework Region 4 108 DVKLVESGGGLLKPGGSLKLSCVAS J Rat Heavy Chain Framework Region 1 109 GFSFSKYW J Heavy Chain CDR1 110 MHWVRQAPGQGLEWIAE J Rat Heavy Chain Framework Region 2 111 IEYDGTET J Heavy Chain CDR2 105 NYAPSIKDRFTISRDNAKNTLYLQMSNVRFE J Rat Heavy Chain Framework Region 3 112 DAATYFC TTEEMHTTDYYYGFAY J Heavy Chain CDR3 113 WGQGTLVTVSS J Rat Heavy Chain Framework Region 4 108 EVQLVESGGGLVQPGRSLKLSCAAS F Rat Heavy Chain Framework Region 1 114 GFIFSEHN F Heavy Chain CDR1 115 MAWVRQAPKKGLEWVAT F Rat Heavy Chain Framework Region 2 116 ISDDGRNT F Heavy Chain CDR2 117 YYRDSMRGRFTISRENARSTLYLQLDSLRSED F Rat Heavy Chain Framework Region 3 118 TATYYC ASHRYNLFDS F Heavy Chain CDR3 119 WGQGVMVTVSS F Rat Heavy Chain Framework Region 4 94 QVQLQQSGTELVKPASSVRISCKAS A Rat Heavy Chain Framework Region 1 120 GYTLTTNY A Heavy Chain CDR1 121 MHWIRQQPGNGLEWIGW A Rat Heavy Chain Framework Region 2 122 IYPGNGNT A Heavy Chain CDR2 123 KFNHKFDGRTTLTADKSSSIVYMQLSSLTSED A Rat Heavy Chain Framework Region 3 124 SAVYFC ARSDFDY A Heavy Chain CDR3 125 WGQGVMVTVSS A Rat Heavy Chain Framework Region 4 94 DVQLEESGGGLVRPGRSLKLSCADS B Rat Heavy Chain Framework Region 1 126 GVNFSNRG B Heavy Chain CDR1 127 MAWVRQAPTKGLEWVAT B Rat Heavy Chain Framework Region 2 128 ISYDGRII B Heavy Chain CDR2 129 YYRDSVKGRFSISRENAKSTLYLQMDSLRSED B Rat Heavy Chain Framework Region 3 130 TATYYC ARHPIAADWYFDF B Heavy Chain CDR3 131 WGPGTMVTVSS B Rat Heavy Chain Framework Region 4 132 EVQLVESGGGSVQPGRSLKLSCAAS I Rat Heavy Chain Framework Region 1 133 GFTFSDYN I Heavy Chain CDR1 134 MAWVRQAPKKGPEWVAT I Rat Heavy Chain Framework Region 2 135 ITYDVHNA I Heavy Chain CDR2 136 YYRDSVKGRFTISRDDAKSTLYLQMDSLRSED I Rat Heavy Chain Framework Region 3 137 TATYFC ARPGAY I Heavy Chain CDR3 138 WGQGTLVTVSS I Rat Heavy Chain Framework Region 4 108 QVRLLQSGAALVKPGASVKMSCKAS O Rat Heavy Chain Framework Region 1 139 GYTFTDYW O Heavy Chain CDR1 140 MSWVKQSHGKSLEWIGE O Rat Heavy Chain Framework Region 2 141 IYPNSGAT O Heavy Chain CDR2 142 NFNEKFKDKATLTVDRSTSTAYMELSRLTSED O Rat Heavy Chain Framework Region 3 143 SAIYYC ARGFPNNYLSWFAY O Heavy Chain CDR3 144 WGQGTLVTVSS O Rat Heavy Chain Framework Region 4 108 QIQLVQSGPELKKPGESVKISCKAS R Rat Heavy Chain Framework Region 1 145 GYTFTNYG R Heavy Chain CDR1 146 MYWVKQAPGQGLQYMGW R Rat Heavy Chain Framework Region 2 147 INTETGKP R Heavy Chain CDR2 148 TYADDFKGRFVFFLETSASTAYLQINNLKNED R Rat Heavy Chain Framework Region 3 149 MATYFC AREVKHGLFHWFAY R Heavy Chain CDR3 150 WGQGTLVTVSS R Rat Heavy Chain Framework Region 4 108 EVQLVESGGGLVQPGRSLTLSCSAS Pi Rat Heavy Chain Framework Region 1 151 GFTFRDYN Pi Heavy Chain CDR1 152 MAWVRQAPRKGLEWVAT Pi Rat Heavy Chain Framework Region 2 153 ISFDDYNT Pi Heavy Chain CDR2 154 YYRDSVKGRFTISRDDAKSTLYLQMDSLRSED Pi Rat Heavy Chain Framework Region 3 155 TATYYC ARPGTY Pi Heavy Chain CDR3 156 WGQGTLVTVSS Pi Rat Heavy Chain Framework Region 4 108 QVQLQQSGAELVKPGSSVRISCKAS Mu Rat Heavy Chain Framework Region 1 157 GYTITSYD Mu Heavy Chain CDR1 158 MHWIKQQPGNGLEGIGW Mu Rat Heavy Chain Framework Region 2 159 IHPGNGKI Mu Heavy Chain CDR2 160 KYNQKFNGKATLTVDKSSSTAYMQLSSLTSE Mu Rat Heavy Chain Framework Region 3 161 DSAVYFC ARGTTRVFPWFAY Mu Heavy Chain CDR3 162 WGQGTLVTVSS Mu Rat Heavy Chain Framework Region 4 108 EVQLVESGGGLVQPGRSLKLSCAAS V Rat Heavy Chain Framework Region 1 114 GFSFSNYG V Heavy Chain CDR1 163 MHWIRQAPTKGLEWVAS V Rat Heavy Chain Framework Region 2 164 ISPTGGNT V Heavy Chain CDR2 165 YYRDSVKGRFTISRDNTKSTLYLQMDSLRSED V Rat Heavy Chain Framework Region 3 166 TATYYC ATDDLYYSGPFAY V Heavy Chain CDR3 167 WGQGTLVTVSS V Rat Heavy Chain Framework Region 4 108 EVQLQESGPGLVKPAQSLSLTCSVTGYSITN G3 Rat Heavy Chain Variable Region 168 MYRWNWIRKFPGNKLEWMGYINTAGSTDY SPSLRGRVSITGDTSKNQFFLHLTSVTTEDTA TYYCAGFITNPFDFWGQGVMVTVSS EVQVVESGGGLVQPGRSLKLSCVPSGFTFNN G5 Rat Heavy Chain Variable Region 169 YWMTWIRQAPGKAPEWVASISNTGGSTFY PDSVRGRFSISRDNTKGTLYLHMTSLRSEDTA TYYCIRNMDAWGQGTSVTVSS GKLVESGGGLLKPGGSLKLSCVASGFTFDKY E7 Rat Heavy Chain Variable Region 170 WMHWVRQAPGKGLEWIAEIEYDGTETNYA PSIKDRFTISRDNAKNTLYLQMSNVRSEDAA TYFCTTEEMYTTDYYYGFAYWGQGTLVTVSS DVKLVESGGGLLKPGGSLKLSCVASGFSFSKY J Rat Heavy Chain Variable Region 171 WMHWVRQAPGQGLEWIAEIEYDGTETNY APSIKDRFTISRDNAKNTLYLQMSNVRFEDA ATYFCTTEEMHTTDYYYGFAYWGQGTLVTV SS EVQLVESGGGLVQPGRSLKLSCAASGFIFSEH F Rat Heavy Chain Variable Region 172 NMAWVRQAPKKGLEWVATISDDGRNTYYR DSMRGRFTISRENARSTLYLQLDSLRSEDTAT YYCASHRYNLFDSWGQGVMVTVSS QVQLQQSGTELVKPASSVRISCKASGYTLTT A Rat Heavy Chain Variable Region 173 NYMHWIRQQPGNGLEWIGWIYPGNGNTK FNHKFDGRTTLTADKSSSIVYMQLSSLTSEDS AVYFCARSDFDYWGQGVMVTVSS DVQLEESGGGLVRPGRSLKLSCADSGVNFSN B Rat Heavy Chain Variable Region 174 RGMAWVRQAPTKGLEWVATISYDGRIIYYR DSVKGRFSISRENAKSTLYLQMDSLRSEDTAT YYCARHPIAADWYFDFWGPGTMVTVSS EVQLVESGGGSVQPGRSLKLSCAASGFTFSD I Rat Heavy Chain Variable Region 175 YNMAWVRQAPKKGPEWVATITYDVHNAYY RDSVKGRFTISRDDAKSTLYLQMDSLRSEDT ATYFCARPGAYWGQGTLVTVSS QVRLLQSGAALVKPGASVKMSCKASGYTFT O Rat Heavy Chain Variable Region 176 DYWMSWVKQSHGKSLEWIGEIYPNSGATN FNEKFKDKATLTVDRSTSTAYMELSRLTSEDS AIYYCARGFPNNYLSWFAYWGQGTLVTVSS QIQLVQSGPELKKPGESVKISCKASGYTFTNY R Rat Heavy Chain Variable Region 177 GMYWVKQAPGQGLQYMGWINTETGKPTY ADDFKGRFVFFLETSASTAYLQINNLKNEDM ATYFCAREVKHGLFHWFAYWGQGTLVTVSS EVQLVESGGGLVQPGRSLTLSCSASGFTFRD Pi Rat Heavy Chain Variable Region 178 YNMAWVRQAPRKGLEWVATISFDDYNTYY RDSVKGRFTISRDDAKSTLYLQMDSLRSEDT ATYYCARPGTYWGQGTLVTVSS QVQLQQSGAELVKPGSSVRISCKASGYTITSY Mu Rat Heavy Chain Variable Region 179 DMHWIKQQPGNGLEGIGWIHPGNGKIKYN QKFNGKATLTVDKSSSTAYMQLSSLTSEDSA VYFCARGTTRVFPWFAYWGQGTLVTVSS EVQLVESGGGLVQPGRSLKLSCAASGFSFSN V Rat Heavy Chain Variable Region 180 YGMHWIRQAPTKGLEWVASISPTGGNTYYR DSVKGRFTISRDNTKSTLYLQMDSLRSEDTAT YYCATDDLYYSGPFAYWGQGTLVTVSS QVQLVQSGAEVKKPGASVKVSCKAS A Humanised 1 Heavy Chain FR 1 181 MHWVRQAPGQRLEWMGW A Humanised 1 Heavy Chain FR 2 182 KFNHKFDGRVTITRDTSASTAYMELSSLRSED A Humanised 1 Heavy Chain FR 3 183 TAVYYC WGQGTLVTVSS A Humanised 1 Heavy Chain FR 4 108 QVQLVQSGAEVKKPGASVKVSCKAS A Humanised 2 Heavy Chain FR 1 181 MHWVRQAPGQGLEWMGW A Humanised 2 Heavy Chain FR 2 184 KFNHKFDGRVTMTRDTSTSTVYMELSSLRSE A Humanised 2 Heavy Chain FR 3 185 DTAVYYC WGQGTMVTVSS A Humanised 2 Heavy Chain FR 4 186 QVQLVQSGAEVKKPGASVKVSCKAS A Humanised 3 Heavy Chain FR 1 181 MHWVRQAPGQGLEWMGW A Humanised 3 Heavy Chain FR 2 184 KFNHKFDGRVTMTRDTSISTAYMELSRLRSD A Humanised 3 Heavy Chain FR 3 187 DTAVYYC WGQGTLVTVSS A Humanised 3 Heavy Chain FR 4 108 QVQLVQSGAEVKKPGSSVKVSCKAS A Humanised 4 Heavy Chain FR 1 188 MHWVRQAPGQGLEWMGW A Humanised 4 Heavy Chain FR 2 184 KFNHKFDGRVTITADKSTSTAYMELSSLRSED A Humanised 4 Heavy Chain FR 3 189 TAVYYC WGQGTLVTVSS A Humanised 4 Heavy Chain FR 4 108 QVQLVQSGSELKKPGASVKVSCKAS A Humanised 5 Heavy Chain FR 1 190 MHWVRQAPGQGLEWMGW A Humanised 5 Heavy Chain FR 2 184 KFNHKFDGRFVFSLDTSVSTAYLQISSLKAED A Humanised 5 Heavy Chain FR 3 191 TAVYYC WGQGTLVTVSS A Humanised 5 Heavy Chain FR 4 108 QVQLVQSGAEVKKPGASVKVSCKASGYTLTT A Humanised 1 Heavy Chain Variable 192 NYMHWVRQAPGQRLEWMGWIYPGNGNT Region KFNHKFDGRVTITRDTSASTAYMELSSLRSED TAVYYCARSDFDYWGQGTLVTVSS QVQLVQSGAEVKKPGASVKVSCKASGYTLTT A Humanised 2 Heavy Chain Variable 193 NYMHWVRQAPGQGLEWMGWIYPGNGNT Region KFNHKFDGRVTMTRDTSTSTVYMELSSLRSE DTAVYYCAISDFDYWGQGTMVTVSS QVQLVQSGAEVKKPGASVKVSCKASGYTLTT A Humanised 3 Heavy Chain Variable 194 NYMHWVRQAPGQGLEWMGWIYPGNGNT Region KFNHKFDGRVTMTRDTSISTAYMELSRLRSD DTAVYYCARSDFDYWGQGTLVTVSS QVQLVQSGAEVKKPGSSVKVSCKASGYTLTT A Humanised 4 Heavy Chain Variable 195 NYMHWVRQAPGQGLEWMGWIYPGNGNT Region KFNHKFDGRVTITADKSTSTAYMELSSLRSED TAVYYCATSDFDYWGQGTLVTVSS QVQLVQSGSELKKPGASVKVSCKASGYTLTT A Humanised 5 Heavy Chain Variable 196 NYMHWVRQAPGQGLEWMGWIYPGNGNT Region KFNHKFDGRFVFSLDTSVSTAYLQISSLKAED TAVYYCARSDFDYWGQGTLVTVSS DIQMTQSPSSLSASVGDRVTITCQAS A Humanised 1 Light Chain FR 1 197 LIWFQQKPGKAPKSLIY A Humanised 1 Light Chain FR 2 198 SLANGVPSRFSGSGSGTDFTLTISSLQPEDFA A Humanised 1 Light Chain FR 3 199 TYYC FGPGTKVDIK A Humanised 1 Light Chain FR 4 200 DIQMTQSPSAMSASVGDRVTITCQAS A Humanised 2 Light Chain FR 1 201 LIWFQQKPGKVPKRLIY A Humanised 2 Light Chain FR 2 202 SLANGVPSRFSGSGSGTEFTLTISSLQPEDFAT A Humanised 2 Light Chain FR 3 203 YYC FGPGTKVDIK A Humanised 2 Light Chain FR 4 200 DIQMTQSPSSLSASVGDRVTITCQAS A Humanised 3 Light Chain FR 1 197 LIWYQQKPGKAPKRLIY A Humanised 3 Light Chain FR 2 204 SLANGVPSRFSGSGSGTEFTLTISSLQPEDFAT A Humanised 3 Light Chain FR 3 203 YYC FGQGTKLEIK A Humanised 3 Light Chain FR 4 205 DIQMTQSPSSLSASVGDRVTITCQAS A Humanised 4 Light Chain FR 1 197 LIWYQQKPGKAPKLLIY A Humanised 4 Light Chain FR 2 206 SLANGVPSRFSGSGSGTDFTFTISSLQPEDIAT A Humanised 4 Light Chain FR 3 207 YYC FGQGTKLEIK A Humanised 4 Light Chain FR 4 205 DIQMTQSPSSVSASVGDRVTITCQAS A Humanised 5 Light Chain FR 1 208 LIWYQQKPGKAPKLLIY A Humanised 5 Light Chain FR 2 206 SLANGVPSRFSGSGSGTDFTLTISSLQPEDFA A Humanised 5 Light Chain FR 3 199 TYYC FGQGTKVEIK A Humanised 5 Light Chain FR 4 209 DIQMTQSPSSLSASVGDRVTITCQASQDIGN A Humanised 1 Light Chain Variable 210 NLIWFQQKPGKAPKSLIYFATSLANGVPSRFS Region GSGSGTDFTLTISSLQPEDFATYYCLQYREYPL TFGPGTKVDIK DIQMTQSPSAMSASVGDRVTITCQASQDIG A Humanised 2 Light Chain Variable 211 NNLIWFQQKPGKVPKRLIYFATSLANGVPSR Region FSGSGSGTEFTLTISSLQPEDFATYYCLQYREY PLTFGPGTKVDIK DIQMTQSPSSLSASVGDRVTITCQASQDIGN A Humanised 3 Light Chain Variable 212 NLIWYQQKPGKAPKRLIYFATSLANGVPSRFS Region GSGSGTEFTLTISSLQPEDFATYYCLQYREYPL TFGQGTKLEIK DIQMTQSPSSLSASVGDRVTITCQASQDIGN A Humanised 4 Light Chain Variable 213 NLIWYQQKPGKAPKLLIYFATSLANGVPSRFS Region GSGSGTDFTFTISSLQPEDIATYYCLQYREYPL TFGQGTKLEIK DIQMTQSPSSVSASVGDRVTITCQASQDIGN A Humanised 5 Light Chain Variable 214 NLIWYQQKPGKAPKLLIYFATSLANGVPSRFS Region GSGSGTDFTLTISSLQPEDFATYYCLQYREYPL TFGQGTKVEIK QVQLVESGGGVVQPGRSLRLSCAAS F Humanised 1 Heavy Chain FR 1 215 MAWVRQAPGKGLEWVAT F Humanised 1 Heavy Chain FR 2 216 YYRDSMRGRFTISRDNSKNTLYLQMNSLRAE F Humanised 1 Heavy Chain FR 3 217 DTAVYYC WGQGTMVTVSS F Humanised 1 Heavy Chain FR 4 186 EVQLVESGGGLVQPGGSLRLSCAAS F Humanised 2 Heavy Chain FR 1 218 MAWVRQAPGKGLEWVST F Humanised 2 Heavy Chain FR 2 219 YYRDSMRGRFTISRDNSKNTLYLQMNSLRAE F Humanised 2 Heavy Chain FR 3 217 DTAVYYC WGQGTLVTVSS F Humanised 2 Heavy Chain FR 4 108 EVQLVESGGGLVQPGGSLRLSCAAS F Humanised 3 Heavy Chain FR 1 218 MAWVRQAPGKGLEWVAT F Humanised 3 Heavy Chain FR 2 216 YYRDSMRGRFTISRDNAKNSLYLQMNSLRAE F Humanised 3 Heavy Chain FR 3 220 DTAVYYC WGQGTMVTVSS F Humanised 3 Heavy Chain FR 4 186 EVQLVESGGGLVQPGGSLRLSCAAS F Humanised 4 Heavy Chain FR 1 218 MAWVRQAPGKGLVWVST F Humanised 4 Heavy Chain FR 2 221 YYRDSMRGRFTISRDNAKNTLYLQMNSLRA F Humanised 4 Heavy Chain FR 3 222 EDTAVYYC WGQGTLVTVSS F Humanised 4 Heavy Chain FR 4 108 EVQLVESGGGLVQPGRSLRLSCAAS F Humanised 5 Heavy Chain FR 1 223 MAWVRQAPGKGLEWVST F Humanised 5 Heavy Chain FR 2 219 YYRDSMRGRFTISRDNAKNSLYLQMNSLRAE F Humanised 5 Heavy Chain FR 3 224 DTALYYC WGQGTLVTVSS F Humanised 5 Heavy Chain FR 4 108 QVQLVESGGGVVQPGRSLRLSCAASGFIFSE F Humanised 1 Heavy Chain Variable 225 HNMAWVRQAPGKGLEWVATISDDGRNTY Region YRDSMRGRFTISRDNSKNTLYLQMNSLRAE DTAVYYCTSHRYNLFDSWGQGTMVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFIFSEH F Humanised 2 Heavy Chain Variable 226 NMAWVRQAPGKGLEWVSTISDDGRNTYYR Region DSMRGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCAKHRYNLFDSWGQGTLVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFIFSEH F Humanised 3 Heavy Chain Variable 227 NMAWVRQAPGKGLEWVATISDDGRNTYY Region RDSMRGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARHRYNLFDSWGQGTMVTVSS EVQLVESGGGLVQPGGSLRLSCAASGFIFSEH F Humanised 4 Heavy Chain Variable 228 NMAWVRQAPGKGLVWVSTISDDGRNTYYR Region DSMRGRFTISRDNAKNTLYLQMNSLRAEDT AVYYCARHRYNLFDSWGQGTLVTVSS EVQLVESGGGLVQPGRSLRLSCAASGFIFSEH F Humanised 5 Heavy Chain Variable 229 NMAWVRQAPGKGLEWVSTISDDGRNTYYR Region DSMRGRFTISRDNAKNSLYLQMNSLRAEDT ALYYCAKHRYNLFDSWGQGTLVTVSS DIQMTQSPSSLSASVGDRVTITCKAS F Humanised 1 Light Chain FR 1 230 LNWYQQKPGKAPKRLIY F Humanised 1 Light Chain FR 2 231 KLQTGVPSRFSGSGSGTEFTLTISSLQPEDFAT F Humanised 1 Light Chain FR 3 232 YYC FGQGTKLEIK F Humanised 1 Light Chain FR 4 205 DIQMTQSPSSLSASVGDRVTITCKAS F Humanised 2 Light Chain FR 1 230 LNWFQQKPGKAPKSLIY F Humanised 2 Light Chain FR 2 233 KLQTGVPSKFSGSGSGTDFTLTISSLQPEDFA F Humanised 2 Light Chain FR 3 234 TYYC FGQGTRLEIK F Humanised 2 Light Chain FR 4 235 DIQMTQSPSSLSASVGDRVTITCKAS F Humanised 3 Light Chain FR 1 230 LNWYQQKPGKAPKLLIY F Humanised 3 Light Chain FR 2 236 KLQTGVPSRFSGSGSGTDFTLTISSLQPEDFA F Humanised 3 Light Chain FR 3 237 TYYC FGQGTKLEIK F Humanised 3 Light Chain FR 4 205 DIQLTQSPSFLSASVGDRVTITCKAS F Humanised 4 Light Chain FR 1 238 LNWYQQKPGKAPKLLIY F Humanised 4 Light Chain FR 2 236 KLQTGVPSRFSGSGSGTEFTLTISSLQPEDFAT F Humanised 4 Light Chain FR 3 232 YYC FGQGTKLEIK F Humanised 4 Light Chain FR 4 205 DIQMTQSPSTLSASVGDRVTITCKAS F Humanised 5 Light Chain FR 1 239 LNWYQQKPGKAPKLLIY F Humanised 5 Light Chain FR 2 236 KLQTGVPSRFSGSGSGTEFTLTISSLQPDDFA F Humanised 5 Light Chain FR 3 240 TYYC FGQGTKLEIK F Humanised 5 Light Chain FR 4 205 DIQMTQSPSSLSASVGDRVTITCKASQNIDR F Humanised 1 Light Chain Variable 241 YLNWYQQKPGKAPKRLIYNTNKLQTGVPSR Region FSGSGSGTEFTLTISSLQPEDFATYYCLQYNSL PLTFGQGTKLEIK DIQMTQSPSSLSASVGDRVTITCKASQNIDR F Humanised 2 Light Chain Variable 242 YLNWFQQKPGKAPKSLIYNTNKLQTGVPSKF Region SGSGSGTDFTLTISSLQPEDFATYYCLQYNSLP LTFGQGTRLEIK DIQMTQSPSSLSASVGDRVTITCKASQNIDR F Humanised 3 Light Chain Variable 243 YLNWYQQKPGKAPKLLIYNTNKLQTGVPSRF Region SGSGSGTDFTLTISSLQPEDFATYYCLQYNSLP LTFGQGTKLEIK DIQLTQSPSFLSASVGDRVTITCKASQNIDRYL F Humanised 4 Light Chain Variable 244 NWYQQKPGKAPKLLIYNTNKLQTGVPSRFS Region GSGSGTEFTLTISSLQPEDFATYYCLQYNSLPL TFGQGTKLEIK DIQMTQSPSTLSASVGDRVTITCKASQNIDR F Humanised 5 Light Chain Variable 245 YLNWYQQKPGKAPKLLIYNTNKLQTGVPSRF Region SGSGSGTEFTLTISSLQPDDFATYYCLQYNSLP LTFGQGTKLEIK ARSDFDY A Humanised 1 Heavy Chain CDR3 125 AISDFDY A Humanised 2 Heavy Chain CDR3 246 ARSDFDY A Humanised 3 Heavy Chain CDR3 125 ATSDFDY A Humanised 4 Heavy Chain CDR3 247 ARSDFDY A Humanised 5 Heavy Chain CDR3 125 AXSDFDY (where X is R, I or T) A General Heavy Chain CDR3 248 TSHRYNLFDS F Humanised 1 Heavy Chain CDR3 249 AKHRYNLFDS F Humanised 2 Heavy Chain CDR3 250 ARHRYNLFDS F Humanised 3 Heavy Chain CDR3 251 ARHRYNLFDS F Humanised 4 Heavy Chain CDR3 251 AKHRYNLFDS F Humanised 5 Heavy Chain CDR3 250 XXHRYNLFDS (where X.sub.1 is A or T and X.sub.2  F General Heavy Chain CDR3 252 is S, K or R) NISSE ROR1 epitope for clone A 253 FQDDL Substituted sequence for epitope 254 mapping

(73) The CDR sequences and the framework regions in the table above have been determined based on information on framework regions and CDRs from the IMGT (the international ImMunoGeneTics information system) database (see www.imgt.org).

(74) An alternative method for labelling CDRs is using the Kabat system and this can give slightly different results. However, this can easily be determined by someone skilled in the art. For the avoidance of doubt, the CDR sequences in the variable regions based on the Kabat system are as follows, where the Kabat CDRs are in bold:

(75) TABLE-US-00004 Clone G3 light chain variable region (SEQ ID NO: 75) DVVMTQTPVSLPVSLGGQVSISCRSSQSLEHSNGDTFLHWYLQKPGQSPRLLIYRVSNRFSGVPDR FSGSGSGTDFTLKISRIEPEDLGDYYCLQSTHFPNTFGAGTKLELK  Clone G5 light chain variable region (SEQ ID NO: 76) DIQLTQSPSTLSASLGERVTISCRASQSISNSLNWYQQKPDGTVKRLIYSTSTLESGVPSRFSGSGSG TDFSLSISSLESEDFAMYYCLQFATYPQVTFGSGTKLEIK  Clone E7 light chain variable region (SEQ ID NO: 77) DIVLTQSPALAVSVGQRATISCRASQSVSISRYNFMHWYQQKPGQQPKLLIYRASNLASGIPARFSG SGSGTDFTLTINPVQADDIATYYCQQNRESPRTFGGGTKLELK  Clone J light chain variable region (SEQ ID NO: 78) DIVLTQSPALAVSVGQRATISCRASQSVSISRYDFMHWYQQKPGQQPKLLIYRASNLASGIPARFSG SGSGTDFTLTINPVQADDIATYYCQQNRESPRTFGGGTKLELK  Clone F light chain variable region (SEQ ID NO: 79) DIQMTQSPSFLSASVGDRVTINCKASQNIDRYLVNYQQKLGEAPKRLLYNTNKLQTGIPSRFSGSG SATDFTLTISSLQPEDFATYFCLQYNSLPLTFGSGTKLEIK  Clone A light chain variable region (SEQ ID NO: 80) DIQMTQSPSSMSASLGDRVTFTCQASQDIGNNLIWFQQKPGKSPRPLMYFATSLANGVPSRFSGSR SGSDYSLTISSLESEDLADYHCLQYREYPLTFGSGTKLDLK  Clone B light chain variable region (SEQ ID NO: 81) DIRMTQSPASLSASLGETVTIECLTSEDIYSDLAWFQQKPGKSPQLLIYDANSLQNGVPSRFGGCGS GTQYSLQISSLQSEDVATYFCQQYKNYPPTFGGGTKLVLK  Clone I light chain variable region (SEQ ID NO: 82) DIQLTQSPSSMSASLGDRVSLTCQSSQGIGKYLSWYQHKPGKPPKAMIYYATKLADGVPSRFSGSR SGSDFSLTISSLESEDIAIYYCLQFDDYPWTFGGGTKLELK  Clone O light chain variable region (SEQ ID NO: 83) DIVLTQSPALAVSLEQRVTIACKTSQNVDNHGISYMHWYQQKSGQEPKLLIYEGSNLAVGIPARFS GSGSGTDFTLTIDPVEADDIETYYCQQSKDDPRTFGGGTKLELK  Clone R light chain variable region (SEQ ID NO: 84) QFTLTQPKSVSGSLRSTITIPCERSSGDIGDSYVSWYQQHLGRPPINVIYADDQRPSEVSDRFSGSIDS SSNSASLTITNLQMDDEADYFCQSYDRNVDFNTVFGGGTKVTVL Clone Pi light chain variable region (SEQ ID NO: 85) DIQLTQSPSSLSASLGDRVSLTCQSSQGIGKYLSWFQHKPGKPPKPVINYATNLADGVPSRFSGRRS GSDFSLTISSLESEDTAIYYCLQFDDFRWTVGGGTKLELK Clone Mu light chain variable region (SEQ ID NO: 86) QFTLTQPKSVSGSLRSTITIPCERSSGDIGDNYVSWYQQHLGRPPINVIYADDQRPSEVSDRFSGSID SSSNSASLTITNLQMDDEADYFCQSFDSNFDIPVFGGGTKLTVL Clone V light chain variable region (SEQ ID NO: 87) DIKMTQSPSFLSASVGDRVTINCKASQNITRFLNWYQQELGEAPTLLIYNTNNLQTGIPSRFSGSGS GTDFTLTISSLQPEDVATYFCLQHGSRPRTFGGGTKLELK Clone G3 heavy chain variable region (SEQ ID NO: 168) EVQLQESGPGLVKPAQSLSLTCSVTGYSITNMYRWNWIRKFPGNKLEWMGYINTAGSTDYSPSLRGRV SITGDTSKNQFFLHLTSVTTEDTATYYCAGFITNPFDFWGQGVMVTVSS Clone G5 heavy chain variable region (SEQ ID NO: 169) EVQVVESGGGLVQPGRSLKLSCVPSGFTFNNYWMTWIRQAPGKAPEWVASISNTGGSTFYPDSVRGRF SISRDNTKGTLYLHMTSLRSEDTATYYCIRNMDAWGQGTSVTVSS Clone E7 heavy chain variable region (SEQ ID NO: 170) GKLVESGGGLLKPGGSLKLSCVASGFTFDKYWMHWVRQAPGKGLEWIAEIEYDGTETNYAPSIKDRF TISRDNAKNTLYLQMSNVRSEDAATYFCTTEEMYTTDYYYGFAYWGQGTLVTVSS Clone J heavy chain variable region (SEQ ID NO: 171) DVKLVESGGGLLKPGGSLKLSCVASGFSFSKYWMHWVRQAPGQGLEWIAEIEYDGTETNYAPSIKDR FTISRDNAKNTLYLQMSNVRFEDAATYFCTTEEMHTTDYYYGFAYWGQGTLVTVSS Clone F heavy chain variable region (SEQ ID NO: 172) EVQLVESGGGLVQPGRSLKLSCAASGFIFSEHNMAWVRQAPKKGLEWVATISDDGRNTYYRDSMRGR FTISRENARSTLYLQLDSLRSEDTATYYCASHRYNLFDSWGQGVMVTVSS Clone Aheavy chain variable region (SEQ ID NO: 173) QVQLQQSGTELVKPASSVRISCKASGYTLTTNYMHWIRQQPGNGLEWIGWIYPGNGNTKFNHKF DGRTTLTADKSSSIVYMQLSSLTSEDSAVYFCARSDFDYWGQGVMVTVSS Clone B heavy chain variable region (SEQ ID NO: 174) DVQLEESGGGLVRPGRSLKLSCADSGVNFSNRGMAWVRQAPTKGLEWVATISYDGRHYYRDSVKGR FSISRENAKSTLYLQMDSLRSEDTATYYCARHPIAADWYFDFWGPGTMVTVSS Clone I heavy chain variable region (SEQ ID NO: 175) EVQLVESGGGSVQPGRSLKLSCAASGFTFSDYNMAWVRQAPKKGPEWVATITYDVHNAYYRDSVKG RFTISRDDAKSTLYLQMDSLRSEDTATYFCARPGAYWGQGTLVTVSS Clone O heavy chain variable region (SEQ ID NO: 176) QVRLLQSGAALVKPGASVKMSCKASGYTFTDYWMSWVKQSHGKSLEWIGEIYPNSGATNFNEKFKD KATLTVDRSTSTAYMELSRLTSEDSAIYYCARGFPNNYLSWFAYWGQGTLVTVSS Clone R heavy chain variable region (SEQ ID NO: 177) QIQLVQSGPELKKPGESVKISCKASGYTFTNYGMYWVKQAPGQGLQYMGWINTETGKPTYADDFKG RFVFFLETSASTAYLQINNLKNEDMATYFCAREVKHGLFHWFAYWGQGTLVTVSS Clone Pi heavy chain variable region (SEQ ID NO: 178) EVQLVESGGGLVQPGRSLTLSCSASGFTFRDYNMAWVRQAPRKGLEWVATISFDDYNTYYRDSVKGR FTISRDDAKSTLYLQMDSLRSEDTATYYCARPGTYWGQGTLVTVSS Clone Mu heavy chain variable region (SEQ ID NO: 179) QVQLQQSGAELVKPGSSVRISCKASGYTITSYDMHWIKQQPGNGLEGIGWIHPGNGKIKYNQKFNGKA TLTVDKSSSTAYMQLSSLTSEDSAVYFCARGTTRVFPWFAYWGQGTLVTVSS Clone V heavy chain variable region (SEQ ID NO: 180) EVQLVESGGGLVQPGRSLKLSCAASGFSFSNYGMHWIRQAPTKGLEWVASISPTGGNTYYRDSVKGRF TISRDNTKSTLYLQMDSLRSEDTATYYCATDDLYYSGPFAYWGQGTLVTVSS Clone A Humanised 1 light chain variable region (SEQ ID NO: 210) DIQMTQSPSSLSASVGDRVTITCQASQDIGNNLIWFQQKPGKAPKSLIYFATSLANGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCLQYREYPLTFGPGTKVDIK Clone A Humanised 2 light chain variable region (SEQ ID NO: 211) DIQMTQSPSAMSASVGDRVTITCQASQDIGNNLIWFQQKPGKVPKRLIYFATSLANGVPSRFSGSG SGTEFTLTISSLQPEDFATYYCLQYREYPLTFGPGTKVDIK Clone A Humanised 3 light chain variable region (SEQ ID NO: 212) DIQMTQSPSSLSASVGDRVTITCQASQDIGNNLIWYQQKPGKAPKRLIYFATSLANGVPSRFSGSGS GTEFTLTISSLQPEDFATYYCLQYREYPLTFGQGTKLEIK Clone A Humanised 4 light chain variable region (SEQ ID NO: 213) DIQMTQSPSSLSASVGDRVTITCQASQDIGNNLIWYQQKPGKAPKLLIYFATSLANGVPSRFSGSGS GTDFTFTISSLQPEDIATYYCLQYREYPLTFGQGTKLEIK Clone A Humanised 5 light chain variable region (SEQ ID NO: 214) DIQMTQSPSSVSASVGDRVTITCQASQDIGNNLIWYQQKPGKAPKLLIYFATSLANGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCLQYREYPLTFGQGTKVEIK Clone A Humanised 1 heavy chain variable region (SEQ ID NO: 192) QVQLVQSGAEVKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQRLEWMGWIYPGNGNTKFNHKFD GRVTITRDTSASTAYMELSSLRSEDTAVYYCARSDFDYWGQGTLVTVSS Clone A Humanised 2 heavy chain variable region (SEQ ID NO: 193) QVQLVQSGAEVKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFD GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAISDFDYWGQGTMVTVSS Clone A Humanised 3 heavy chain variable region (SEQ ID NO: 194) QVQLVQSGAEVKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFD GRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSDFDYWGQGTLVTVSS Clone A Humanised 4 heavy chain variable region (SEQ ID NO: 195) QVQLVQSGAEVKKPGSSVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFD GRVTITADKSTSTAYMELSSLRSEDTAVYYCATSDFDYWGQGTLVTVSS Clone A Humanised 5 heavy chain variable region (SEQ ID NO: 196) QVQLVQSGSELKKPGASVKVSCKASGYTLTTNYMHWVRQAPGQGLEWMGWIYPGNGNTKFNHKFD GRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARSDFDYWGQGTLVTVSS Clone F Humanised 1 light chain variable region (SEQ ID NO: 241) DIQMTQSPSSLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKRLIYNTNKLQTGVPSRFSGSG SGTEFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 2 light chain variable region (SEQ ID NO: 242) DIQMTQSPSSLSASVGDRVTITCKASQNIDRYLNWFQQKPGKAPKSLIYNTNKLQTGVPSKFSGSG SGTDFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTRLEIK Clone F Humanised 3 light chain variable region (SEQ ID NO: 243) DIQMTQSPSSLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 4 light chain variable region (SEQ ID NO: 244) DIQLTQSPSFLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFSGSG SGTEFTLTISSLQPEDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 5 light chain variable region (SEQ ID NO: 245) DIQMTQSPSTLSASVGDRVTITCKASQNIDRYLNWYQQKPGKAPKLLIYNTNKLQTGVPSRFSGSG SGTEFTLTISSLQPDDFATYYCLQYNSLPLTFGQGTKLEIK Clone F Humanised 1 heavy chain variable region (SEQ ID NO: 225) QVQLVESGGGVVQPGRSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVATISDDGRNTYYRDSMRG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCTSHRYNLFDSWGQGTMVTVSS Clone F Humanised 2 heavy chain variable region (SEQ ID NO: 226) EVQLVESGGGLVQPGGSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVSTISDDGRNTYYRDSMRGR FTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHRYNLFDSWGQGTLVTVSS Clone F Humanised 3 heavy chain variable region (SEQ ID NO: 227) EVQLVESGGGLVQPGGSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVATISDDGRNTYYRDSMRGR FTISRDNAKNSLYLQMNSLRAEDTAVYYCARHRYNLFDSWGQGTMVTVSS Clone F Humanised 4 heavy chain variable region (SEQ ID NO: 228) EVQLVESGGGLVQPGGSLRLSCAASGFIFSEHNMAWVRQAPGKGLVWVSTISDDGRNTYYRDSMRGR FTISRDNAKNTLYLQMNSLRAEDTAVYYCARHRYNLFDSWGQGTLVTVSS Clone F Humanised 5 heavy chain variable region (SEQ ID NO: 229) EVQLVESGGGLVQPGRSLRLSCAASGFIFSEHNMAWVRQAPGKGLEWVSTISDDGRNTYYRDSMRGR FTISRDNAKNSLYLQMNSLRAEDTALYYCAKHRYNLFDSWGQGTLVTVSS

(76) Therefore, the CDRs when determined using the Kabat system are as follows:

(77) TABLE-US-00005 SEQ ID Sequence Clone Description NO: RSSQSLEHSNGDTFLH G3 Light Chain CDR1 255 RVSNRFS G3 Light Chain CDR2 256 LQSTHFPNT G3 Light Chain CDR3 6 NMYRWN G3 Heavy Chain CDR1 257 YINTAGSTDYSPSLRG G3 Heavy Chain CDR2 258 FITNPFDF G3 Heavy Chain CDR3 259 RASQSISNSLN G5 Light Chain CDR1 260 STSTLES G5 Light Chain CDR2 261 LQFATYPQVT G5 Light Chain CDR3 13 NYWMT G5 Heavy Chain CDR1 262 SISNTGGSTFYPDSVRG G5 Heavy Chain CDR2 263 NMDA G5 Heavy Chain CDR3 264 RASQSVSISRYNFMH E7 Light Chain CDR1 265 RASNLAS E7 Light Chain CDR2 266 QQNRESPRT E7 Light Chain CDR3 20 KYWMH E7 Heavy Chain CDR1 267 EIEYDGTETNYAPSIKD E7 Heavy Chain CDR2 268 EEMYTTDYYYGFAY E7 Heavy Chain CDR3 269 RASQSVSISRYDFMH J Light Chain CDR1 270 RASNLAS J Light Chain CDR2 266 QQNRESPRT J Light Chain CDR3 20 KYWMH J Heavy Chain CDR1 267 EIEYDGTETNYAPSIKD J Heavy Chain CDR2 268 EEMHTTDYYYGFAY J Heavy Chain CDR3 271 KASQNIDRYLN F Light Chain CDR1 272 NTNKLQT F Light Chain CDR2 273 LQYNSLPLT F Light Chain CDR3 28 EHNMA F Heavy Chain CDR1 274 TISDDGRNTYYRDSMRG F Heavy Chain CDR2 275 HRYNLFDS F Heavy Chain CDR3 276 QASQDIGNNLI A Light Chain CDR1 277 FATSLAN A Light Chain CDR2 278 LQYREYPLT A Light Chain CDR3 34 TNYMH A Heavy Chain CDR1 279 WIYPGNGNTKFNHKFDG A Heavy Chain CDR2 280 SDFDY A Heavy Chain CDR3 281 LTSEDIYSDLA B Light Chain CDR1 282 DANSLQN B Light Chain CDR2 283 QQYKNYPPT B Light Chain CDR3 41 NRGMA B Heavy Chain CDR1 284 TISYDGRIIYYRDSVKG B Heavy Chain CDR2 285 HPIAADWYFDF B Heavy Chain CDR3 286 QSSQGIGKYLS I Light Chain CDR1 287 YATKLAD I Light Chain CDR2 288 LQFDDYPWT I Light Chain CDR3 48 DYNMA I Heavy Chain CDR1 289 TITYDVHNAYYRDSVKG I Heavy Chain CDR2 290 PGAY I Heavy Chain CDR3 291 KTSQNVDNHGISYMH O Light Chain CDR1 292 EGSNLAV O Light Chain CDR2 293 QQSKDDPRT O Light Chain CDR3 54 DYWMS O Heavy Chain CDR1 294 EIYPNSGATNFNEKFKD O Heavy Chain CDR2 295 GFPNNYLSWFAY O Heavy Chain CDR3 296 ERSSGDIGDSYVS R Light Chain CDR1 297 ADDQRPS R Light Chain CDR2 298 QSYDRNVDFNTV R Light Chain CDR3 60 NYGMY R Heavy Chain CDR1 299 WINTETGKPTYADDFKG R Heavy Chain CDR2 300 EVKHGLFHWFAY R Heavy Chain CDR3 301 QSSQGIGKYLS Pi Light Chain CDR1 287 YATNLAD Pi Light Chain CDR2 302 LQFDDFRWT Pi Light Chain CDR3 65 DYNMA Pi Heavy Chain CDR1 289 TISFDDYNTYYRDSVKG Pi Heavy Chain CDR2 303 PGTY Pi Heavy Chain CDR3 304 ERSSGDIGDNYV Mu Light Chain CDR1 305 ADDQRPS Mu Light Chain CDR2 298 QSFDSNFDIPV Mu Light Chain CDR3 68 SYDMH Mu Heavy Chain CDR1 306 WIHPGNGKIKYNQKFNG Mu Heavy Chain CDR2 307 GTTRVFPWFAY Mu Heavy Chain CDR3 308 KASQNITRFLN V Light Chain CDR1 309 NTNNLQT V Light Chain CDR2 310 LQHGSRPRT V Light Chain CDR3 74 NYGMH V Heavy Chain CDR1 311 SISPTGGNTYYRDSVKG V Heavy Chain CDR2 312 DDLYYSGPFAY V Heavy Chain CDR3 313 QASQDIGNNLI Humanised A Light Chain CDR1 277 FATSLAN Humanised A Light Chain CDR2 278 LQYREYPLT Humanised A Light Chain CDR3 34 TNYMH Humanised A Heavy Chain CDR1 279 WIYPGNGNTKFNHKFDG Humanised A Heavy Chain CDR2 280 SDFDY Humanised A Heavy Chain CDR3 281 KASQNIDRYLN Humanised F Light Chain CDR1 272 NTNKLQT Humanised F Light Chain CDR2 273 LQYNSLPLT Humanised F Light Chain CDR3 28 EHNMA Humanised F Heavy Chain CDR1 274 TISDDGRNTYYRDSMRG Humanised F Heavy Chain CDR2 275 HRYNLFDS Humanised F Heavy Chain CDR3 276