Antibody constructs for EGFRVIII and CD3

Abstract

The present invention relates to a bispecific antibody construct comprising a first binding domain which binds to human EGFRVIII on the surface of a target cell and a second binding domain which binds to human CD3 on the surface of a T cell. Moreover, the invention provides a polynucleotide encoding the antibody construct, a vector comprising said polynucleotide and a host cell transformed or transfected with said polynucleotide or vector. Furthermore, the invention provides a process for the production of the antibody construct of the invention, a medical use of said antibody construct and a kit comprising said antibody construct.

Claims

1. A bispecific antibody construct comprising a first binding domain which binds to human and macaque epidermal growth factor receptor VIII (EGFRVIII) on the surface of a target cell and a second binding domain which binds to human CD3 on the surface of a T cell, wherein the first binding domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 157 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 158.

2. The antibody construct according to claim 1, wherein the antibody construct is in a format selected from the group consisting of (scFv).sub.2, diabodies, and oligomers thereof.

3. The antibody construct according to claim 1, wherein the second binding domain binds to human CD3 epsilon and to Callithrix jacchus, Saguinus oedipus or Saimiri sciureus CD3 epsilon, and comprises: (i) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 11, a CDR-L2 comprising the amino acid of SEQ ID NO:12, and a CDR-L3 comprising the amino acid of SEQ ID NO:13; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 14, a CDR-H2 comprising the amino acid of SEQ ID NO:15, and a CDR-H3 comprising the amino acid of SEQ ID NO:16; (ii) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 20, a CDR-L2 comprising the amino acid of SEQ ID NO:21, and a CDR-L3 comprising the amino acid of SEQ ID NO:22; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 23, a CDR-H2 comprising the amino acid of SEQ ID NO:24, and a CDR-H3 comprising the amino acid of SEQ ID NO:25; (iii) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 29, a CDR-L2 comprising the amino acid of SEQ ID NO:30, and a CDR-L3 comprising the amino acid of SEQ ID NO:31; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 32, a CDR-H2 comprising the amino acid of SEQ ID NO:33, and a CDR-H3 comprising the amino acid of SEQ ID NO:34; (iv) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 38, a CDR-L2 comprising the amino acid of SEQ ID NO:39, and a CDR-L3 comprising the amino acid of SEQ ID NO:40; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 41, a CDR-H2 comprising the amino acid of SEQ ID NO:42, and a CDR-H3 comprising the amino acid of SEQ ID NO:43; (v) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 47, a CDR-L2 comprising the amino acid of SEQ ID NO:48, and a CDR-L3 comprising the amino acid of SEQ ID NO:49; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 50, a CDR-H2 comprising the amino acid of SEQ ID NO:51, and a CDR-H3 comprising the amino acid of SEQ ID NO:52; (vi) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 56, a CDR-L2 comprising the amino acid of SEQ ID NO:57, and a CDR-L3 comprising the amino acid of SEQ ID NO:58; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 59, a CDR-H2 comprising the amino acid of SEQ ID NO:60, and a CDR-H3 comprising the amino acid of SEQ ID NO:61; (vii) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 65, a CDR-L2 comprising the amino acid of SEQ ID NO:66, and a CDR-L3 comprising the amino acid of SEQ ID NO:67; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 68, a CDR-H2 comprising the amino acid of SEQ ID NO:69, and a CDR-H3 comprising the amino acid of SEQ ID NO:70; (viii) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 74, a CDR-L2 comprising the amino acid of SEQ ID NO:75, and a CDR-L3 comprising the amino acid of SEQ ID NO:76; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 77, a CDR-H2 comprising the amino acid of SEQ ID NO:78, and a CDR-H3 comprising the amino acid of SEQ ID NO:79; (ix) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 83, a CDR-L2 comprising the amino acid of SEQ ID NO:84, and a CDR-L3 comprising the amino acid of SEQ ID NO:85; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 86, a CDR-H2 comprising the amino acid of SEQ ID NO:87, and a CDR-H3 comprising the amino acid of SEQ ID NO:88; (x) a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 92, a CDR-L2 comprising the amino acid of SEQ ID NO:93, and a CDR-L3 comprising the amino acid of SEQ ID NO:94; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 95, a CDR-H2 comprising the amino acid of SEQ ID NO:96, and a CDR-H3 comprising the amino acid of SEQ ID NO:97; or (xi) a VH region comprising amino acid sequence of SEQ ID NO: 101, and a VL region comprising amino acid sequence of SEQ ID NO: 102.

4. The antibody construct according to claim 1, comprising: (a) a polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 159; (ii) a peptide linker comprising the amino acid sequence selected from any one of SEQ ID NOs: 1-9; (iii) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and (iv) optionally, a His-tag comprising the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide comprising in following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 159; (ii) a peptide linker comprising the amino acid sequence selected from any one of SEQ ID NOs: 1-9; (iii) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; (iv) optionally, a peptide linker comprising the amino acid sequence selected from any one of SEQ ID NOs: 1-9; (v) a polypeptide comprising the amino acid sequence of SEQ ID NO:134; and (vi) optionally, a His-tag comprising the amino acid sequence of SEQ ID NO: 10; (c) a polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence QRFVTGHFGGLX.sub.1PANG (SEQ ID NO: 135) whereas X.sub.1 is Y or H; (ii) a polypeptide comprising the amino acid sequence of: SEQ ID NO: 159; (iii) a peptide linker comprising the amino acid sequence selected from any one of SEQ ID NOs: 1-9; (iv) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; (v) a polypeptide comprising the amino acid sequence QRFVTGHFGGLHPANG (SEQ ID NO: 137) or QRFCTGHFGGLHPCNG (SEQ ID NO: 139); and (vi) optionally, a His-tag comprising the amino acid sequence of SEQ ID NO: 10; (d) a first polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98, and SEQ ID NO: 101; (ii) a peptide linker comprising the amino acid sequence of SEQ ID NO: 8; (iii) a polypeptide comprising the amino acid of SEQ ID NO: 158; and (iv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 140; and a second polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 157; (ii) a peptide linker comprising the amino acid sequence of SEQ ID NO: 8; (iii) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, and SEQ ID NO: 102, and a serine residue at the C-terminus; and (iv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 141; (e) a first polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98, and SEQ ID NO: 101; (ii) a peptide linker comprising the amino acid sequence of SEQ ID NO: 8; (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 158; and (iv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 142; and a second polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 157; (ii) a peptide linker comprising the amino acid sequence of SEQ ID NO: 8; (iii) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99, and SEQ ID NO: 102, and a serine residue at the C-terminus; and (iv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 143; (f) a first polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 159; (ii) a peptide linker comprising the amino acid sequence selected from any one of SEQ ID NOs: 1-9; (iii) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and (iv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 144; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 145; (g) a first polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 159; and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 146; and a second polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 147; (h) a first polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 159; and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 148; and a second polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and (ii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 149; (i) a polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 159; (ii) a peptide linker comprising the amino acid sequence selected from any one of SEQ ID NOs: 1-9; (iii) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and (iv) a polypeptide comprising the amino acid sequence of SEQ ID NO: 150; or (j) a polypeptide comprising in the following order starting from the N-terminus: (i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 159; (ii) a peptide linker comprising the amino acid sequence selected from any one of SEQ ID NOs: 1-9; (iii) a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; (iv) a peptide linker having an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1, 2, 4, 5, 6, 8 and 9; and (v) the third domain comprising the amino acid sequence selected from any one of SEQ ID NOs: 181-188.

5. The antibody construct according to claim 4, comprising a polypeptide comprising the amino acid sequence of SEQ ID NO: 160.

6. A polynucleotide encoding the antibody construct according to claim 1.

7. A vector comprising the polynucleotide according to claim 6.

8. An isolated host cell transformed or transfected with the polynucleotide according to claim 6.

9. A process for producing the antibody construct according to claim 1, said process comprising culturing a host cell comprising a polynucleotide encoding a bispecific antibody construct comprising a first binding domain which binds to human and macaque EGFRVIII on the surface of a target cell and a second binding domain which binds to human CD3 on the surface of a T cell, wherein the first binding domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 157 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 158, under conditions allowing the expression of the antibody construct, and recovering the produced antibody construct from the culture.

10. A pharmaceutical composition comprising the antibody construct according to claim 1 and a carrier, stabilizer, excipient, diluent, solubilizer, surfactant, emulsifier, preservative or adjuvant.

11. A polypeptide comprising: (a) a first binding domain that binds to human and macaque Epidermal Growth Factor receptor VIII (EGFRVIII), and comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 157, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 158; and (b) a second binding domain that binds to human CD3, and comprises: a VL region comprising: a CDR-L1 comprising the amino acid of SEQ ID NO: 92, a CDR-L2 comprising the amino acid of SEQ ID NO:93, and a CDR-L3 comprising the amino acid of SEQ ID NO:94; and a VH region comprising: a CDR-H1 comprising the amino acid of SEQ ID NO: 95, a CDR-H2 comprising the amino acid of SEQ ID NO:96, and a CDR-H3 comprising the amino acid of SEQ ID NO:97.

12. The polypeptide of claim 11, comprising (a) a first binding domain that binds to human and macaque EGFRVIII, and comprises: a VH comprising the amino acid sequence of SEQ ID NO: 157, and a VL comprising the amino acid sequence of SEQ ID NO: 158; and (b) a second binding domain which binds to human CD3, and comprises a VH comprising the amino acid sequence of SEQ ID NO: 98, and a VL comprising the amino acid sequence of SEQ ID NO: 99.

13. The polypeptide of claim 11, comprising (a) a first domain comprising the amino acid sequence as set forth in SEQ ID NO:159; and (b) a second domain comprising the amino acid sequence as set forth in SEQ ID NO:100.

14. The polypeptide according to claim 13, further comprising a His-tag comprising the amino acid sequence of SEQ ID NO: 10.

15. The polypeptide according to claim 13, comprising the amino acid sequence as set forth in SEQ ID NO:189.

16. The polypeptide according to claim 13, comprising the amino acid sequence as set forth in SEQ ID NO:190.

17. A polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 160.

18. The polypeptide of claim 17, further comprising a His-tag comprising the amino acid sequence of SEQ ID NO: 10.

19. The polypeptide of claim 17, comprising in the following order starting from the N-terminus: an amino acid sequence as set forth in SEQ ID NO: 160, and a His-tag as set forth in SEQ ID NO: 10.

20. The polypeptide of claim 17, further comprising a single-chain Fc (scFc) comprising an amino acid sequence selected from any one of SEQ Nos. 181-188.

21. The polypeptide of claim 20, comprising the amino acid sequence as set forth in SEQ ID NO:189.

22. The polypeptide of claim 20, comprising the amino acid sequence as set forth in SEQ ID NO:190.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

(1) FIG. 1:

(2) Schematic representation of EGFRvIII, which is a tumor-specific EGFR mutant found in glioblastoma with a deletion of 267 amino acids at the N-terminus.

(3) FIG. 2

(4) Sequence comparison of the target binder EvIII-2 (SEQ ID NO: 169) and its derivative with covalently connected V-Regions EvIII-1 (SEQ ID NO: 159).

(5) FIG. 3

(6) Purification of EvIII-1 (FIG. 3B) and EvIII-2 (FIG. 3A) following standard research scale production

(7) FIG. 4

(8) EvIII-1 and EvIII-2 monomer: Reducing SDS-PAGE

(9) FIG. 5

(10) Cross-Reactivity of EvIII-1 and EvIII-2 bispecific antibody constructs shown by Flowcytometry:

(11) Binding to human and macaque EGFRvIII and CD3

(12) FIG. 6

(13) Binding of EvIII-1 and EvIII-2 bispecific antibody constructs to EGFRvIII transfected cells as well as human glioblastoma cell line U87

(14) FIG. 7

(15) Cytotoxic activity of stimulated human CD8 T cells against human EGFRvIII-transfected CHO cells. 18-hour 51chromium release assay. Effector cells: stimulated enriched human CD8 T cells. Target cells: EGFRvIII transfected CHO cells. Effector to target cell (E:T) ratio: 10:1

(16) FIG. 8

(17) Cytotoxic activity of stimulated human CD8 T cells against the human gliomablastoma cell line U87: (a) 18-hour 51chromium release assay. Effector cells: stimulated enriched human CD8 T cells. Target cells: vIII high positive U87 cells. Effector to target cell (E:T) ratio: 10:1; (b) 18-hour FACS based assay. Effector cells: stimulated enriched human CD8 T cells. Target cells: hu EGFRvIII high positive U87 glioma cells. Effector to target cell (E:T) ratio: 10:1.

(18) FIG. 9

(19) Cytotoxic activity of stimulated human CD8 T cells against a human tumor cell line expressing native EGFRvIII Antigen: glioblastoma cell line DK-MG: 18-hour 51chromium release assay. Effector cells: stimulated enriched human CD8 T cells. Target cells: DK-MG cells. Effector to target cell (E:T) ratio: 10:1.

(20) FIG. 10

(21) Cytotoxic activity of a macaque T cell line against macaque EGFRvIII-transfected CHO cells: 48-hour FACS-based cytotoxicity assay. Effector cells: macaque CD3+ LnPx4119. Target cells: macaque EGFRvIII transfected CHO cells. Effector to target cell (E:T)-ratio: 10:1

(22) FIG. 11

(23) Stability of the bispecific antibody constructs after incubation for 24 Hours in human plasma:

(24) 18-hour 51Cr based assay. Effector cells: stimulated enriched human CD8 T cells. Target cells: hu EGFRvIII transfected CHO cells. Effector to target cell (E:T) ratio: 10:1. antibody constructs as indicated

(25) FIG. 12

(26) The protein homogeneity of the EGFRvIII antibody constructs analyzed High Resolution Cation Exchange Chromatography CIEX

(27) FIG. 13

(28) The surface hydrophobicity of bispecific antibody constructs tested in Hydrophobic Interaction Chromatography HIC in flow-through mode

(29) FIG. 14

(30) Monomer to dimer conversion after 7 days of incubation at 250 g/ml and 37 C. Analyzed with HP-SEC.

(31) FIG. 15

(32) Monomer to dimer conversion after three freeze/thaw cycles at 250 g/ml and 37 C. Analyzed with HP-SEC.

(33) FIG. 16:

(34) FACS binding analysis of EvIII-1xCD3-scFc construct to CHO cells transfected with the human EGFR as well as human CD3+ T cell line HPBaLL. The red line represents cells incubated with 2 g/ml purified monomeric protein that are subsequently incubated with the mouse anti-I2C antibody and the PE labeled goat anti mouse IgG detection antibody. The black histogram line reflects the negative control: cells only incubated with the anti-I2C antibody as well as the PE labeled detection antibody

(35) FIG. 17:

(36) Cytotoxic activity induced by EvIII-1xCD3-scFc construct redirected to CD56 depleted unstimulated human PBMCs as effector cells and CHO cells transfected with human EGFR as target cells. (Example 1.2)

EXAMPLES

(37) The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The present invention is limited only by the claims.

Example 1

(38) Cytotoxic Activity

(39) The potency of EGFRVIIIxCD3 bispecific antibody constructs of the invention in redirecting effector T cells against EGFRVIII-expressing target cells was analyzed in five in vitro cytotoxicity assays: The potency of EGFRVIIxCD3 bispecific antibody constructs in redirecting stimulated human CD8+ effector T cells against human EGFRVIII-transfected CHO cells was measured in an 18 hour .sup.51Cr release assay (Effector target ration 10:1). FIG. 7 The potency of EGFRVIIxCD3 bispecific antibody constructs in redirecting stimulated human CD8+ effector T cells against the EGFRVIII positive human glioblastoma cell line U87 was measured in an 18 hour .sup.51Cr release assay (Effector target ration 10:1). FIG. 8 The potency of EGFRVIIxCD3 bispecific antibody constructs in redirecting the T cells in unstimulated human PBMC (CD14-/CD56-) against human EGFRVIII-transfected CHO cells in the absence and presence of soluble EGFRVIII was measured in a 48 hour FACS-based cytotoxicity assay (Effector target ration 10:1). FIG. 6 and Table 6 The potency of EGFRVIIxCD3 bispecific antibody constructs in redirecting the T cells in unstimulated human PBMC (CD14.sup./CD56.sup.) against the EGFRVIII-positive human glioblastoma cell line U87 was measured in a 48 hour FACS-based cytotoxicity assay. FIG. 8 For confirmation that the cross-reactive EGFRVIIIxCD3 bispecific antibody constructs are capable of redirecting macaque T cells against macaque EGFRVIII-transfected CHO cells, a 48 hour FACS-based cytotoxicity assay was performed with a macaque T cell line LnPx4119 as effector T cells (Effector target ration 10:1). FIG. 10

Example 1.1

(40) Chromium Release Assay with Stimulated Human T Cells

(41) Stimulated T cells enriched for CD8+ T cells were obtained as described in the following. A petri dish (145 mm diameter, Greiner bio-one GmbH, Kremsmnster) was coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) in a final concentration of 1 g/ml for 1 hour at 37 C. Unbound protein was removed by one washing step with PBS. 3-5107 human PBMC were added to the precoated petri dish in 120 ml of RPMI 1640 with stabilized glutamine/10% FCS/IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2 days. On the third day, the cells were collected and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and the cells were cultured again for one day in the same cell culture medium as above. CD8+ cytotoxic T lymphocytes (CTLs) were enriched by depletion of CD4+ T cells and CD56+ NK cells using Dynal-Beads according to the manufacturer's protocol.

(42) Cyno EGFRVIII- or human EGFRVIII-transfected CHO target cells were washed twice with PBS and labeled with 11.1 MBq 51Cr in a final volume of 100 l RPMI with 50% FCS for 60 minutes at 37 C. Subsequently, the labeled target cells were washed 3 times with 5 ml RPMI and then used in the cytotoxicity assay. The assay was performed in a 96-well plate in a total volume of 200 l supplemented RPMI with an E:T ratio of 10:1. A starting concentration of 0.01-1 g/ml of purified bispecific antibody construct and threefold dilutions thereof were used. Incubation time for the assay was 18 hours. Cytotoxicity was determined as relative values of released chromium in the supernatant relative to the difference of maximum lysis (addition of Triton-X) and spontaneous lysis (without effector cells). All measurements were carried out in quadruplicates. Measurement of chromium activity in the supernatants was performed in a Wizard 3 gamma counter (Perkin Elmer Life Sciences GmbH, Kln, Germany). Analysis of the results was carried out with Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, Calif., USA). EC50 values calculated by the analysis program from the sigmoidal dose response curves were used for comparison of cytotoxic activity.

Example 1.2

(43) Potency of Redirecting Stimulated Human Effector T Cells Against Human EGFRVIII-Transfected CHO Cells

(44) The cytotoxic activity of EGFRVIIxCD3 bispecific antibody constructs according to the invention was analyzed in a 51-chromium (51Cr) release cytotoxicity assay using CHO cells transfected with human EGFRVIII as target cells, and stimulated human CD8+ T cells as effector cells. The experiment was carried out as described in Example 1.1.

(45) The EGFRVIIxCD3 bispecific antibody constructs showed very potent cytotoxic activity against human EGFRVIII transfected CHO cells in the 1-digit picomolar range.

Example 1.3

(46) Potency of Redirecting Stimulated Human Effector T Cells Against the EGFRVIII Positive Human Cell Line Human Glioblastoma Cell Line DK-MG

(47) The cytotoxic activity of EGFRVIIxCD3 bispecific antibody constructs was analyzed in a 51-chromium (51Cr) release cytotoxicity assay using the EGFRVIII-positive human glioblastoma cell line DK-MG as source of target cells, and stimulated human CD8+ T cells as effector cells. The assay was carried out as described in Example 1.1.

(48) In accordance with the results of the 51-chromium release assays with stimulated enriched human CD8+ T lymphocytes as effector cells and human EGFRVIII-transfected CHO cells as target cells, EGFRVIIxCD3 bispecific antibody constructs of the present invention are also potent in cytotoxic activity against natural expresser target cells; see FIG. 9.

Example 1.4

(49) FACS-Based Cytotoxicity Assay with Unstimulated Human PBMC

(50) Isolation of Effector Cells

(51) Human peripheral blood mononuclear cells (PBMC) were prepared by Ficoll density gradient centrifugation from enriched lymphocyte preparations (buffy coats), a side product of blood banks collecting blood for transfusions. Buffy coats were supplied by a local blood bank and PBMC were prepared on the same day of blood collection. After Ficoll density centrifugation and extensive washes with Dulbecco's PBS (Gibco), remaining erythrocytes were removed from PBMC via incubation with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 100 M EDTA). Platelets were removed via the supernatant upon centrifugation of PBMC at 100g. Remaining lymphocytes mainly encompass B and T lymphocytes, NK cells and monocytes. PBMC were kept in culture at 37 C./5% CO2 in RPMI medium (Gibco) with 10% FCS (Gibco).

(52) Depletion of CD14+ and CD56+ Cells

(53) For depletion of CD14+ cells, human CD14 MicroBeads (Milteny Biotec, MACS, #130-050-201) were used, for depletion of NK cells human CD56 MicroBeads (MACS, #130-050-401). PBMC were counted and centrifuged for 10 min at room temperature with 300g. The supernatant was discarded and the cell pellet resuspended in MACS isolation buffer [80 L/107 cells; PBS (Invitrogen, #20012-043), 0.5% (v/v) FBS (Gibco, #10270-106), 2 mM EDTA (Sigma-Aldrich, #E-6511)]. CD14 MicroBeads and CD56 MicroBeads (20 L/107 cells) were added and incubated for 15 min at 4-8 C. The cells were washed with MACS isolation buffer (1-2 mL/107 cells). After centrifugation (see above), supernatant was discarded and cells resuspended in MACS isolation buffer (500 L/108 cells). CD14/CD56 negative cells were then isolated using LS Columns (Miltenyi Biotec, #130-042-401). PBMC w/o CD14+/CD56+ cells were cultured in RPMI complete medium i.e. RPMI1640 (Biochrom AG, #FG1215) supplemented with 10% FBS (Biochrom AG, #S0115), 1 non-essential amino acids (Biochrom AG, #K0293), 10 mM Hepes buffer (Biochrom AG, #L1613), 1 mM sodium pyruvate (Biochrom AG, #L0473) and 100 U/mL penicillin/streptomycin (Biochrom AG, #A2213) at 37 C. in an incubator until needed.

(54) Target Cell Labeling

(55) For the analysis of cell lysis in flow cytometry assays, the fluorescent membrane dye DiOC18 (DiO) (Molecular Probes, #V22886) was used to label human EGFRVIII- or macaque EGFRVIII-transfected CHO cells as target cells and distinguish them from effector cells. Briefly, cells were harvested, washed once with PBS and adjusted to 106 cell/mL in PBS containing 2% (v/v) FBS and the membrane dye DiO (5 L/106 cells). After incubation for 3 min at 37 C., cells were washed twice in complete RPMI medium and the cell number adjusted to 1.25105 cells/mL. The vitality of cells was determined using 0.5% (v/v) isotonic EosinG solution (Roth, #45380).

(56) Flow Cytometry Based Analysis

(57) This assay was designed to quantify the lysis of cyno or human EGFRVIII-transfected CHO cells in the presence of serial dilutions of EGFRVIII bispecific antibody constructs. Equal volumes of DiO-labeled target cells and effector cells (i.e., PBMC w/o CD14+ cells) were mixed, resulting in an E:T cell ratio of 10:1. 160 l of this suspension were transferred to each well of a 96-well plate. 40 L of serial dilutions of the EGFRVIIxCD3 bispecific antibody constructs and a negative control bispecific (a CD3-based bispecific antibody construct recognizing an irrelevant target antigen) or RPMI complete medium as an additional negative control were added. The bispecific antibody-mediated cytotoxic reaction proceeded for 48 hours in a 7% CO2 humidified incubator. Then cells were transferred to a new 96-well plate and loss of target cell membrane integrity was monitored by adding propidium iodide (PI) at a final concentration of 1 g/mL. PI is a membrane impermeable dye that normally is excluded from viable cells, whereas dead cells take it up and become identifiable by fluorescent emission.

(58) Samples were measured by flow cytometry on a FACSCanto II instrument and analyzed by FACSDiva software (both from Becton Dickinson). Target cells were identified as DiO-positive cells. PI-negative target cells were classified as living target cells. Percentage of cytotoxicity was calculated according to the following formula:

(59) $\mathrm{Cytotoxicity}\left[\%\right]=\frac{n_{\mathrm{dead}\mathrm{target}\mathrm{cells}}}{n_{\mathrm{target}\mathrm{cells}}}100$$n=\mathrm{number}\mathrm{of}\mathrm{events}$

(60) Using GraphPad Prism 5 software (Graph Pad Software, San Diego), the percentage of cytotoxicity was plotted against the corresponding bispecific antibody construct concentrations. Dose response curves were analyzed with the four parametric logistic regression models for evaluation of sigmoid dose response curves with fixed hill slope and EC50 values were calculated.

Example 1.5

(61) Potency of Redirecting Unstimulated Human PBMC Against Human EGFRVIII-Transfected CHO Cells in Absence and Presence of Soluble EGFRVIII

(62) The cytotoxic activity of EGFRVIIIxCD3 bispecific antibody constructs was analyzed in a FACS-based cytotoxicity assay using CHO cells transfected with human EGFRVIII as target cells, and unstimulated human PBMC as effector cells. The assay was carried out as described in Example 1.4 above.

(63) Expectedly, EC50 values were generally higher in cytotoxicity assays with unstimulated PBMC as effector cells compared with cytotoxicity assays using stimulated human CD8+ T cells (see Example 1.2).

Example 1.6

(64) Potency of Redirecting Unstimulated Human PBMC Against the EGFRVIII-Positive Human Glioblastoma Cell Line U87 or DK-MG Cells

(65) The cytotoxic activity of EGFRVIIxCD3 bispecific antibody constructs was furthermore analyzed in a FACS-based cytotoxicity assay using the EGFRVIII-positive human glioblastoma cell line U87 or DK-MG as a source of target cells and unstimulated human PBMC as effector cells. The assay was carried out as described in Example 1.4 above. The results are shown in FIGS. 8 and 9.

Example 1.7

(66) Potency of Redirecting Macaque T Cells Against Macaque EGFRVIII-Expressing CHO Cells

(67) The cytotoxic activity of EGFRVIIxCD3 bispecific antibody constructs was analyzed in a FACS-based cytotoxicity assay using CHO cells transfected with macaque (cyno) EGFRVIII as target cells, and the macaque T cell line 4119LnPx (Knappe et al. Blood 95:3256-61 (2000)) as source of effector cells. Target cell labeling of macaque EGFRVIII-transfected CHO cells and 51Cr-release based analysis of cytotoxic activity was performed as described above.

(68) Results are shown in FIG. 10. Macaque T cells from cell line 4119LnPx were induced to efficiently kill macaque EGFRVIII-transfected CHO cells by EGFRVIIxCD3 bispecific antibody constructs of the invention.

Example 1.8

(69) Potency Gap Between the Monomeric and the Dimeric Isoform of Bispecific Antibody Constructs

(70) In order to determine the difference in cytotoxic activity between the monomeric and the dimeric isoform of individual EGFRVIIxCD3 bispecific antibody constructs (referred to as potency gap), an 18 hour 51-chromium release cytotoxicity assay was carried out as described hereinabove (Example 1.1) with purified bispecific antibody construct monomer and dimer. Effector cells were stimulated enriched human CD8+ T cells. Target cells were hu EGFRVIII transfected CHO cells. Effector to target cell (E:T) ratio was 10:1. The potency gap was calculated as ratio between EC50 values.

Example 2

(71) Stability after Incubation for 24 Hours in Human Plasma

(72) Purified bispecific antibody constructs were incubated at a ratio of 1:5 in a human plasma pool at 37 C. for 96 hours at a final concentration of 2-20 g/ml. After plasma incubation the antibody constructs were compared in a 51-chromium release assay with stimulated enriched human CD8+ T cells and human EGFRVIII-transfected CHO cells at a starting concentration of 0.01-0.1 g/ml and with an effector to target cell (E:T) ratio of 10:1 (assay as described in Example 1.1). Non-incubated, freshly thawed bispecific antibody constructs were included as controls.

(73) The results are shown in FIG. 11; The antibody construct EvIII-2 had a plasma stability (EC50 plasma/EC50 control) of around 2. Surprisingly, the bispecific antibody construct of the invention did show almost no conversion.

Example 3

(74) Protein Homogeneity by High Resolution Cation Exchange Chromatography

(75) The protein homogeneity the antibody constructs of the invention was analyzed by high resolution cation exchange chromatography CIEX.

(76) 50 g of antibody construct monomer were diluted with 50 ml binding buffer A (20 mM sodium dihydrogen phosphate, 30 mM NaCl, 0.01% sodium octanate, pH 5.5), and 40 ml of this solution were applied to a 1 ml BioPro SP-F column (YMC, Germany) connected to an Akta Micro FPLC device (GE Healthcare, Germany). After sample binding, a wash step with further binding buffer was carried out. For protein elution, a linear increasing salt gradient using buffer B (20 mM sodium dihydrogen phosphate, 1000 mM NaCl, 0.01% sodium octanate, pH 5.5) up to 50% percent buffer B was applied over 10 column volumes. The whole run was monitored at 280, 254 and 210 nm optical absorbance. Analysis was done by peak integration of the 280 nm signal recorded in the Akta Unicorn software run evaluation sheet.

(77) The results are shown in FIG. 12. Almost all tested antibody constructs have a very favourable homogeneity of 95% (area under the curve (=AUC) of the main peak).

Example 4

(78) Surface Hydrophobicity as Measured by HIC Butyl

(79) The surface hydrophobicity of bispecific antibody constructs of the invention was tested in Hydrophobic Interaction Chromatography HIC in flow-through mode.

(80) 50 g of antibody construct monomer were diluted with generic formulation buffer to a final volume of 500 l (10 mM citric acid, 75 mM lysine HCl, 4% trehalose, pH 7.0) and applied to a 1 ml Butyl Sepharose FF column (GE Healthcare, Germany) connected to a Akta Purifier FPLC system (GE Healthcare, Germany). The whole run was monitored at 280, 254 and 210 nm optical absorbance. Analysis was done by peak integration of the 280 nm signal recorded in the kta Unicorn software run evaluation sheet. Elution behavior was evaluated by comparing area and velocity of rise and decline of protein signal thereby indicating the strength of interaction of the BiTE albumin fusion with the matrix.

(81) The antibody constructs had a good elution behaviour, which was mostly rapid and complete; see FIG. 13.

Example 5

(82) Monomer to Dimer Conversion after (i) Three Freeze/Thaw Cycles and (ii) 7 Days of Incubation at 250 g/ml

(83) Bispecific EGFRVIIxCD3 antibody monomeric construct were subjected to different stress conditions followed by high performance SEC to determine the percentage of initially monomeric antibody construct, which had been converted into dimeric antibody construct.

(84) (i) 25 g of monomeric antibody construct were adjusted to a concentration of 250 g/ml with generic formulation buffer and then frozen at 80 C. for 30 min followed by thawing for 30 min at room temperature. After three freeze/thaw cycles the dimer content was determined by HP-SEC.
(ii) 25 g of monomeric antibody construct were adjusted to a concentration of 250 g/ml with generic formulation buffer followed by incubation at 37 C. for 7 days. The dimer content was determined by HP-SEC.

(85) A high resolution SEC Column TSK Gel G3000 SWXL (Tosoh, Tokyo-Japan) was connected to an Akta Purifier 10 FPLC (GE Lifesciences) equipped with an A905 Autosampler. Column equilibration and running buffer consisted of 100 mM KH2PO4200 mM Na2SO4 adjusted to pH 6.6. The antibody solution (25 g protein) was applied to the equilibrated column and elution was carried out at a flow rate of 0.75 ml/min at a maximum pressure of 7 MPa. The whole run was monitored at 280, 254 and 210 nm optical absorbance. Analysis was done by peak integration of the 210 nm signal recorded in the Akta Unicorn software run evaluation sheet. Dimer content was calculated by dividing the area of the dimer peak by the total area of monomer plus dimer peak.

(86) The results are shown in FIGS. 14 and 15 below. The EVIII-1xCD3 bispecific antibody constructs of the invention presented with dimer percentages of 0.59% after three freeze/thaw cycles (FIG. 15), and with dimer percentages of 0.26% after 7 days of incubation at 37 C. while the EvIII-1xCD3 bispecific antibody construct showed higher values of 1.56% after 7 days and 2.53% after three freeze/thaw cycles.

Example 6

(87) Thermostability

(88) Antibody aggregation temperature was determined as follows: 40 l of antibody construct solution at 250 g/ml were transferred into a single use cuvette and placed in a Wyatt Dynamic Light Scattering device DynaPro Nanostar (Wyatt). The sample was heated from 40 C. to 70 C. at a heating rate of 0.5 C./min with constant acquisition of the measured radius. Increase of radius indicating melting of the protein and aggregation was used by the software package delivered with the DLS device to calculate the aggregation temperature of the antibody construct.

(89) TABLE-US-00002 TABLE 2 Thermostability of the bispecific antibody constructs as determined by DLS (dynamic light scattering) Thermal Stability EGFRVIII HALB BiTE DLS T.sub.A [ C.] EvIII-1 51.6 EvIII-2 51.2

Example 7

(90) Turbidity at 2500 g/ml Antibody Concentration

(91) 1 ml of purified antibody construct solution of a concentration of 250 g/ml was concentrated by spin concentration units to 2500 g/ml. After 16 h storage at 5 C. the turbidity of the antibody solution was determined by OD340 nm optical absorption measurement against the generic formulation buffer.

(92) The results are shown in Table 3 below. While the EvIII-1 antibody construct of the invention had an extreme favourable turbidity of 0.03 the EvIII-2 antibody construct showed significant turbidity, which is indicative for less favourable characteristics in the formulation of such molecule in a pharmaceutical composition.

(93) TABLE-US-00003 TABLE 3 Turbidity of the antibody constructs after concentration to 2.5 mg/ml over night Turbidity after 16 h EGFRVIII HALB BiTE @ 2500 g/ml [OD340] EvIII-1 0.029 EvIII-2 2.87

(94) TABLE-US-00004 TABLE4 SequenceListing SEQ ID Descrip- NO: tion Source Sequence 1 Peptide artifi- GGGG linker cial 2 Peptide artifi- GGGGS linker cial 3 Peptide artifi- GGGGQ linker cial 4 Peptide artifi- PGGGGS linker cial 5 Peptide artifi- PGGDGS linker cial 6 Peptide artifi- SGGGGS linker cial 7 Peptide artifi- GGGGSGGGS linker cial 8 Peptide artifi- GGGGSGGGGS linker cial 9 Peptide artifi- GGGGSGGGGSGGGGS linker cial 10 Hexa- artifi- HHHHHH histidine cial 11 CDR-L1 artifi- GSSTGAVTSGYYPN ofF6A cial 12 CDR-L2 artifi- GTKFLAP ofF6A cial 13 CDR-L3 artifi- ALWYSNRWV ofF6A cial 14 CDR-H1 artifi- IYAMN ofF6A cial 15 CDR-H2 artifi- RIRSKYNNYATYYADSVKS ofF6A cial 16 CDR-H3 artifi- HGNFGNSYVSFFAY ofF6A cial 17 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNT F6A cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSS 18 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV F6A cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 19 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNT ofF6A cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 20 CDR-L1 artifi- GSSTGAVTSGYYPN ofH2C cial 21 CDR-L2 artifi- GTKFLAP ofH2C cial 22 CDR-L3 artifi- ALWYSNRWV ofH2C cial 23 CDR-H1 artifi- KYAMN ofH2C cial 24 CDR-H2 artifi- RIRSKYNNYATYYADSVKD ofH2C cial 25 CDR-H3 artifi- HGNFGNSYISYWAY ofH2C cial 26 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT H2C cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 27 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV H2C cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 28 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT ofH2C cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 29 CDR-L1 artifi- GSSTGAVTSGYYPN ofH1E cial 30 CDR-L2 artifi- GTKFLAP ofH1E cial 31 CDR-L3 artifi- ALWYSNRWV ofH1E cial 32 CDR-H1 artifi- SYAMN ofH1E cial 33 CDR-H2 artifi- RIRSKYNNYATYYADSVKG ofH1E cial 34 CDR-H3 artifi- HGNFGNSYLSFWAY ofH1E cial 35 VHof artifi- EVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT H1E cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSS 36 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV H1E cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 37 VH-VL artifi- EVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT ofH1E cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 38 CDR-L1 artifi- GSSTGAVTSGYYPN ofG4H cial 39 CDR-L2 artifi- GTKFLAP ofG4H cial 40 CDR-L3 artifi- ALWYSNRWV ofG4H cial 41 CDR-H1 artifi- RYAMN ofG4H cial 42 CDR-H2 artifi- RIRSKYNNYATYYADSVKG ofG4H cial 43 CDR-H3 artifi- HGNFGNSYLSYFAY ofG4H cial 44 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT G4H cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSS 45 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV G4H cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 46 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT ofG4H cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 47 CDR-L1 artifi- RSSTGAVTSGYYPN ofA2J cial 48 CDR-L2 artifi- ATDMRPS ofA2J cial 49 CDR-L3 artifi- ALWYSNRWV ofA2J cial 50 CDR-H1 artifi- VYAMN ofA2J cial 51 CDR-H2 artifi- RIRSKYNNYATYYADSVKK ofA2J cial 52 CDR-H3 artifi- HGNFGNSYLSWWAY ofA2J cial 53 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNT A2J cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSS 54 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGV A2J cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 55 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNT ofA2J cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 56 CDR-L1 artifi- GSSTGAVTSGYYPN ofE1L cial 57 CDR-L2 artifi- GTKFLAP ofE1L cial 58 CDR-L3 artifi- ALWYSNRWV ofE1L cial 59 CDR-H1 artifi- KYAMN ofE1L cial 60 CDR-H2 artifi- RIRSKYNNYATYYADSVKS ofE1L cial 61 CDR-H3 artifi- HGNFGNSYTSYYAY ofE1L cial 62 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNT E1L cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSS 63 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV E1L cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 64 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNT ofE1L cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 65 CDR-L1 artifi- RSSTGAVTSGYYPN ofE2M cial 66 CDR-L2 artifi- ATDMRPS ofE2M cial 67 CDR-L3 artifi- ALWYSNRWV ofE2M cial 68 CDR-H1 artifi- GYAMN ofE2M cial 69 CDR-H2 artifi- RIRSKYNNYATYYADSVKE ofE2M cial 70 CDR-H3 artifi- HRNFGNSYLSWFAY ofE2M cial 71 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNT E2M cial AYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSS 72 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGV E2M cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 73 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNT ofE2M cial AYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 74 CDR-L1 artifi- GSSTGAVTSGYYPN ofF7O cial 75 CDR-L2 artifi- GTKFLAP ofF7O cial 76 CDR-L3 artifi- ALWYSNRWV ofF7O cial 77 CDR-H1 artifi- VYAMN ofF7O cial 78 CDR-H2 artifi- RIRSKYNNYATYYADSVKK ofF7O cial 79 CDR-H3 artifi- HGNFGNSYISWWAY ofF7O cial 80 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNT F7O cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSS 81 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV F7O cial QPEDEAEYYCALWYSNRWVFGGGTKLTVL 82 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNT ofF7O cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVF GGGTKLTVL 83 CDR-L1 artifi- GSSTGAVTSGNYPN ofF12Q cial 84 CDR-L2 artifi- GTKFLAP ofF12Q cial 85 CDR-L3 artifi- VLWYSNRWV ofF12Q cial 86 CDR-H1 artifi- SYAMN ofF12Q cial 87 CDR-H2 artifi- RIRSKYNNYATYYADSVKG ofF12Q cial 88 CDR-H3 artifi- HGNFGNSYVSWWAY ofF12Q cial 89 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT F12Q cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS 90 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV F12Q cial QPEDEAEYYCVLWYSNRWVFGGGTKLTVL 91 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT ofF12Q cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVL 92 CDR-L1 artifi- GSSTGAVTSGNYPN ofI2C cial 93 CDR-L2 artifi- GTKFLAP ofI2C cial 94 CDR-L3 artifi- VLWYSNRWV ofI2C cial 95 CDR-H1 artifi- KYAMN ofI2C cial 96 CDR-H2 artifi- RIRSKYNNYATYYADSVKD ofI2C cial 97 CDR-H3 artifi- HGNFGNSYISYWAY ofI2C cial 98 VHof artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT I2C cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 99 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV I2C cial QPEDEAEYYCVLWYSNRWVFGGGTKLTVL 100 VH-VL artifi- EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT ofI2C cial AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVL 101 VHof artifi- EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT F12q cial AYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS 102 VLof artifi- QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGV F12q cial QPEDEAEYYCVLWYSNRWVFGGGTKLTVL 103 F12q scFv EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNT AYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVF GGGTKLTVL 104 HALB human DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL 105 HALB7 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL 106 HALB098 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL 107 HALB114 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPHLVAASKAALGL 108 HALB254 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALGVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 109 HALB253 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 110 HALB131 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPHLVAASQAALGL 111 HALB135 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPHLVAASKAALGL 112 HALB133 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASKAALGL 113 HALB234 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPKLVAASKAALGL 114 HALBC34S artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL 115 HALB7C34S artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL 116 HALB098 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 117 HALB114 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPHLVAASKAALGL 118 HALB254 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALGVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 119 HALB253 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 120 HALB131 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPHLVAASQAALGL 121 HALB135 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPHLVAASKAALGL 122 HALB133 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASKAALGL 123 HALB234 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34S cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPKLVAASKAALGL 124 HALBC34A artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL 125 HALB7C34A artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAGTFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAAMDDFAAFVEKCCK ADDKETCFAEEGKKLVAASQAALGL 126 HALB098 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 127 HALB114 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPHLVAASKAALGL 128 HALB254 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALGVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 129 HALB253 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASQAALGL 130 HALB131 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPHLVAASQAALGL 131 HALB135 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPHLVAASKAALGL 132 HALB133 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDKFAAFVEKCCK ADDKETCFAEEGPKLVAASKAALGL 133 HALB234 artifi- DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATL C34A cial RETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYA EAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGE YKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTES LVNRRPCFSALDVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPKLVAASKAALGL 134 Ab156 artifi- RDWDFDVFGGGTPVGG cial 135 linear artifi- QRFVTGHFGGLXPANG FcRn cial binding peptide 136 linear artifi- QRFVTGHFGGLYPANG FcRn cial binding peptide Y 137 linear artifi- QRFVTGHFGGLHPANG FcRn cial binding peptide H 138 core artifi- TGHFGGLHP FcRn cial binding peptide H 139 cyclic artifi- QRFCTGHFGGLHPCNG FcRn cial binding peptide H 140 Cross ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT body1 YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW HC YVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 141 Cross GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK body1 SHRSYSCQVTHEGSTVEKTVAPTECSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK LC FNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 142 Cross ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQT body2 YTCNVDHKPSNTKVDKTVEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW HC YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK 143 Cross GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWK body2 SHRSYSCQVTHEGSTVEKTVAPTECSEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE LC DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK 144 Hetero- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTY Fc RCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVE binder WESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc 145 Hetero- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTY Fc RCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE partner WESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc 146 Maxibody EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQ 1 YGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPS target DIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc 147 Maxibody EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQ 1 YGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS CD3Fc DIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 148 Maxibody EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 2 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPS target DIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc 149 Maxibody EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ 2 YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS CD3Fc DIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 150 Mono APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH Fc QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 151 EvIII-1 VHCDR1 NYGMH 152 EvIII-1 VHCDR2 VIWYDGSDKYYADSVRG 153 EvIII-1 VHCDR3 DGYDILTGNPRDFDY 154 EvIII-1 VLCDR1 RSSQSLVHSDGNTYLS 155 EvIII-1 VLCDR2 RISRRFS 156 EvIII-1 VLCDR3 MQSTHVPRT 157 EvIII-1 VH QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSS 158 EvIII-1 VL DTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEI SRVEAEDVGVYYCMQSTHVPRTFGCGTKVEIIK 159 EvIII-1 scFv QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASI SCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPR TFGCGTKVEIK 160 EvIII-1 bi- QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY specific LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASI molecule SCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPR TFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVL 161 EvIII-2 VHCDR1 NYGMH 162 EvIII-2 VHCDR2 VIWYDGSDKYYADSVRG 163 EvIII-2 VHCDR3 DGYDILTGNPRDFDY 164 EvIII-2 VLCDR1 RSSQSLVHSDGNTYLS 165 EvIII-2 VLCDR2 RISRRFS 166 EvIII-2 VLCDR3 MQSTHVPRT 167 EvIII-2 VH QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKGLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSS 168 EvIII-2 VL DTVMTQTPLSSHVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEI SRVEAEDVGVYYCMQSTHVPRTFGQGTKVEIK 169 EvIII-2 scFv QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKGLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASI SCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPR TFGQGTKVEIK 170 EvIII-2 bi- QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKGLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY specific LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASI molecule SCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPR TFGQGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVL 171 EGFR human LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKH vIII FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEI IRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIIS NRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCH PECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTY GCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLT PSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEIL DEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNY LEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQ SDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKF RELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPS TSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQ SVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGS HQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA 172 EGFR cyno- LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDTLSINATNIKH vIII molgus FKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEI IRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSSQKTKIIS NRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCQNVSRGRECVDKCNILEGEPREFVENSECIQCH PECLPQVMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTY GCTGPGLEGCARNGPKIPSIATGMLGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLT PSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEIL DEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNY LEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQ SDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKF RELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPS TSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQ SVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGS HQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA 173 Fc artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA monomer-1 cial KTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS +c/g REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 174 Fcmonomer- artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 2 cial KTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS +c/g/delGK REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP 175 Fcmonomer- artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 3 cial KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS c/+g REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 176 Fcmonomer- artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 4 cial KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS c/+g/delGK REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP 177 Fcmonomer- artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 5 cial KTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS c/g REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 178 Fcmonomer- artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 6 cial KTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS c/g/delGK REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP 179 Fcmonomer- artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 7 cial KTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS +c/+g REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK 180 Fcmonomer- artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA 8 cial KTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS +c/+g/delGK REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSP 181 scFc-1 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGS TYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 182 scFc-2 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTY RCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSP 183 scFc-3 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 184 scFc-4 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSP 185 scFc-5 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 186 scFc-6 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSP 187 scFc-7 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYNS TYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK 188 scFc-8 artifi- DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA cial KTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYNSTY RCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSP 189 EvIII- Bi- QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY 1xCD3- specific LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASI scFc HLE SCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPR molecule TFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 190 EvIII- Bi- QVQLVESGGGVVQSGRSLRLSCAASGFTFRNYGMHWVRQAPGKCLEWVAVIWYDGSDKYYADSVRGRFTISRDNSKNTLY 1xCD3- specific LQMNSLRAEDTAVYYCARDGYDILTGNPRDFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDTVMTQTPLSSHVTLGQPASI scFc_ HLE SCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYRISRRFSGVPDRFSGSGAGTDFTLEISRVEAEDVGVYYCMQSTHVPR delGK molecule TFGCGTKVEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYA DSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVLGGGGDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 191 Peptide (G.sub.4S).sub.4 GGGGSGGGGSGGGGSGGGGS linker linker 192 Peptide (G.sub.4S).sub.5 GGGGSGGGGSGGGGSGGGGSGGGGS linker linker 193 Peptide (G.sub.4S).sub.6 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker linker 194 Peptide (G.sub.4S).sub.7 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker linker 195 Peptide (G.sub.4S).sub.8 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS linker linker