LONG LIFE POLYPEPTIDE BINDING MOLECULES

Abstract

The present invention relates to a binding molecule comprising at least three domains comprised in at least one polypeptide chain, wherein the first binding domain is a binding domain which is capable of binding to a cell surface molecule on a target cell, the second binding domain is a binding domain which is capable of binding to the T cell CD3 receptor complex, and the third domain is a binding domain which is capable of binding to serum albumin, wherein said third domain is positioned at the C-terminus of said second domain. Moreover, the invention provides a nucleic acid sequence encoding the binding molecule, a vector comprising said nucleic acid sequence and a host cell transformed or transfected with said vector. Furthermore, the invention provides a process for the production of the binding molecule of the invention, a medical use of said binding molecule and a kit comprising said binding molecule.

Claims

1. A binding molecule comprising at least three binding domains comprised in at least one polypeptide chain, wherein (a) the first domain is a binding domain which is capable of binding to a cell surface molecule on a target cell; and (b) the second domain is a binding domain which is capable of binding to the T cell CD3 receptor complex; and (c) the third domain is a binding domain which is capable of binding to serum albumin, wherein said third domain is positioned at the C-terminus of said second domain.

2. The binding molecule according to claim 1, wherein the three domains are on one polypeptide in the order from the N-terminus to the C-terminus the first binding domain; the second binding domain; and the third binding domain.

3. The binding molecule according to claim 1, wherein the third binding domain of the binding molecule is an scFv or a single domain antibody.

4. The binding molecule according to claim 1, wherein (a) the first binding domain is capable of binding to the cell surface molecule on a human and a non-human primate cell; (b) the second binding domain is capable of binding to the T cell CD3 receptor complex on a human and a non-human primate cell, and (c) the third binding domain is capable of binding to human and non-human primate serum albumin.

5. The binding molecule according to claim 1, wherein the third binding domain capable of binding to serum albumin is derived from a combinatorial library or an antibody binding domain.

6. The binding molecule according to claim 1, wherein the third binding domain comprises between 10 and 25 aa amino acid residues.

7. The binding molecule according to claim 1, wherein the third binding domain capable of binding to serum albumin comprises the amino acid sequence Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp (SEQ ID NO: 102), wherein Xaa is any amino acid.

8. The binding molecule according to claim 1, wherein the third binding domain capable of binding to serum albumin is derived from a CDR of a single domain antibody.

9. The binding molecule according to claim 1, wherein the third binding domain is binding to serum albumin with an affinity (KD) of <500 nM.

10. The binding molecule according to claim 1, wherein the binding molecule shows cytotoxic activity in an in vitro assay measuring the lysis of target cells by effector cells in the presence of 10% human serum albumin.

11. The binding molecule according to claim 1, wherein the molecule consists of a single polypeptide chain.

12. The binding molecule according to claim 1, wherein (a) the first binding domain comprises an antibody derived VL and VH chain; and/or (b) the second binding domain comprises an antibody derived VL and VH chain.

13. The binding molecule according to claim 1, wherein the molecule comprises one or more further heterologous polypeptide.

14. The binding molecule according to claim 1, wherein the first-binding domain capable of binding to a cell surface molecule is binding to a tumor antigen.

15. The binding molecule according to claim 1, wherein the second binding domain capable of binding to the T cell CD3 receptor complex is capable of binding-to an epitope of human and Callithrix jacchus, Saguinus oedipus or Saimiri sciureus CD3ε chain, wherein the epitope is part of an amino acid sequence of SEQ ID NO: 2, 4, 6, or 8 and comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (SEQ ID NO: 103).

16. The binding molecule according to claim 1, characterized by the amino acid sequence set forth in SEQ ID NO: 51, 52, 54, 55, 57, 58, 60, 61, 75, 76, 81, 82, 85, 86, 90, 91, 85, 96, 100 or 101.

17-20. (canceled)

21. A pharmaceutical composition comprising the binding molecule of claim 1, or produced according to the process of claim 20.

22. (canceled)

23. A method for treating or ameliorating a disease selected from the group consisting of a proliferative disease, an inflammatory disease, an infectious disease and an autoimmune disease, comprising the step of administering to a subject in need thereof the binding molecule of claim 1.

24. A kit comprising the binding molecule of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0245] FIGS. 1A-1C: FACS binding analysis of designated bispecific antibody constructs to CHO cells expressing human PSMA as described in Example 1, and the human CD3+ T cell line HPB-ALL, respectively. The FACS staining was performed as described in Example 1. The bold (dark) lines represent cells incubated with cell culture supernatant of transfected cells expressing the bispecific antibody constructs. The light (grey) histograms show the negative controls. Supernatant of untransfected CHO cells was used as negative control.

[0246] FIGS. 2A-2B: The figures show results of chromium release assays measuring cytotoxic activity induced by designated bispecific antibody constructs redirected to CHO cell line transfected with human PSMA. As effector cells stimulated human CD 8+ T cells were used. The assays was performed in RPMI 1640 supplemented with 10% HSA and is described in Example 2.

[0247] FIG. 3: Cytotoxicity analysis of EpCAM BiTE molecules in the unmodified form or as fusion with SA21 or with HSA. Dose response curves were generated with addition of FCS, without addition of FCS, with addition 10% human serum and with addition of 20% human serum.

[0248] FIG. 4: Cytotoxicity analysis of EpCAM BiTE molecules in the unmodified form or as fusion with SA21 or with HSA. Dose response curves were generated with addition of 8 g/l HSA, with addition of 20 g/l HSA, with addition 10% human serum and with addition of 20% human serum.

[0249] FIGS. 5A-5F: The diagrams of FIGS. 5A-5F show results of FACS based cytotoxicity assays measuring cytotoxic activity induced by designated bispecific antibody constructs redirected to CHO cell line transfected with HIV gp140. As effector cells stimulated human CD 8.sup.+ T cells were used. The assays was performed in RPMI 1640 supplemented with 10% FCS (5A, 5C, and 5E) or 50% Human Serum (5B, 5D, and 5F) respectively and is described in Example 9.

[0250] FIGS. 6A-6C: HSA/MSA Biacore experiment (surface plasmon resonance) Diagrams show affinity analyses of designated bispecific antibody constructs to immobilized HSA or MSA respectively. The Biacore experiments were performed as described in Example 10. The diagrams show the association and dissociation characteristics of designated bispecific constructs at different concentrations.

[0251] FIG. 7: human CD3 Biacore experiment (surface plasmon resonance) Affinity analyses of designated bispecific antibody constructs to immobilized human CD3. The Biacore experiments were performed as described in Example 11. The diagrams show the association and dissociation characteristics of designated bispecific constructs at different concentrations.

[0252] FIG. 8: Affinity analyses of designated bispecific antibody constructs to immobilized human CD3. The Biacore experiments were performed as described in Example 12. The diagrams show the association and dissociation characteristics of designated bispecific constructs at different concentrations.

[0253] FIG. 9: SEC profile of CD4(1+2)×aCD3L×SA21 The diagram shows the size exclusion profile of CD4(1+2)×aCD3L×SA21. Peaks of aggregate, multimeric and monomer forms are indicated. The blue line indicates optical absorption at 280 nm. The purification method is described in Example 13.

EXAMPLES

[0254] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration, and the present invention is limited only by the claims.

Example 1: Bispecific Binding and Interspecies Cross-Reactivity

[0255] For flow cytometry human PSMA-transfected CHO target cells (the general procedure for the transfection of CHO cells with cell surface molecules to create appropriate target cells is derivable from WO 2008/119567) were used. 200,000 cells of the respective cell lines were incubated for 30 min on ice with 50 μl of purified bispecific molecules at a concentration of 5 pg/ml. The cells were washed twice in PBS with 2% FCS and binding of the constructs was detected with a murine PentaHis antibody (Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS). After washing, bound PentaHis antibodies were detected with an Fc gamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted 1:100 in PBS with 2% FCS.

[0256] FIG. 1 shows the histograms of those FACS analyses. The results with respect to the detection of binding to the target PSMA and to CD3 are summarized in the appended tables 3 and 4. The parental variants of the identified BiTE molecules without an ABP are described in detail, including specific examples for the amino acid sequence, in WO 2010/037836.

TABLE-US-00003 TABLE 3 Binding characteristics of PSMA BiTE antibody molecules with ABP at the N-terminus PSMA CD3 Construct binding binding AB01 × PSMA × CD3 − + AB14 × PSMA × CD3 − + AB156 × PSMA × CD3 − + DX236 × PSMA × CD3 − + DX321 × PSMA × CD3 − + SA04 × PSMA × CD3 − + SA08 × PSMA × CD3 − + SA21 × PSMA × CD3 − + SA25 × PSMA × CD3 − +

TABLE-US-00004 TABLE 4 Binding characteristics of PSMA BiTE antibody molecules with ABP at the C-terminus PSMA CD3 Construct binding binding PSMA × CD3 × AB01 + + PSMA × CD3 × AB14 + + PSMA × CD3 × AB156 + + PSMA × CD3 × DX236 + + PSMA × CD3 × DX321 + + PSMA × CD3 × SA04 + + PSMA × CD3 × SA08 + + PSMA × CD3 × SA21 + + PSMA × CD3 × SA25 + +

Example 2: Cytotoxic Activity

[0257] Chromium Release Assay with Stimulated Human T Cells

[0258] Stimulated T cells enriched for CD8.sup.+ T cells were obtained as described below.

[0259] A petri dish (145 mm diameter, Greiner bio-one GmbH, Kremsmünster) was coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) in a final concentration of 1 μg/ml for 1 hour at 37° C. Unbound protein was removed by one washing step with PBS. 3-5×10.sup.7 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.

[0260] CD8.sup.+ 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.

[0261] Macaque or human PSMA-transfected CHO target cells (the general procedure for the transfection of CHO cells with cell surface molecules to create appropriate target cells is derivable from WO 2008/119567) were washed twice with PBS and labeled with 11.1 MBq 51Cr in a final volume of 100 μl 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 μl supplemented RPMI (as above) with an E:T ratio of 10:1. A starting concentration of 0.01-1 μg/ml of purified bispecific antibody 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″ gammacounter (Perkin Elmer Life Sciences GmbH, Köln, Germany). Analysis of the results was carried out with Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, Calif., USA). EC.sub.50 values calculated by the analysis program from the sigmoidal dose response curves were used for comparison of cytotoxic activity.

[0262] The results for the kill of PSMA expressing target cells are shown in FIG. 2.

Example 3: Biacore-Based Determination of BiTE-Antibody Affinity to Serum Albumin of Different Species

[0263] To confirm binding of the tagged PSMA BiTE molecules to human serum albumin and serum albumins of other species, the following serum albumin preparations were provided: Human serum albumin (HSA), Sigma A9511; Rhesus monkey serum albumin (RSA), bioWorld 22070099; Mouse serum albumin (MSA), Sigma A3139; Bovine serum albumin (BSA), Sigma A7906.

[0264] Binding experiments were performed using plasmon resonance measurements in a Biacore machine. In detail, CM5 Sensor Chips (GE Healthcare) were immobilized with the respective serum albumin protein using acetate buffer pH 4.5 according to the manufacturer's manual. The BiTE antibody samples were loaded in five relevant concentrations e.g.: 50 nM, 25 nM, 12.5 nM, 6.25 nM and 3.13 nM diluted in HBS-EP running buffer (GE Healthcare). For affinity determinations the flow rate was 35 μl/min for 3 min, then HBS-EP running buffer was applied for 10 min again at a flow rate of 35 μl/ml. Regeneration of the chip was performed using a buffer consisting of 100 mM glycine 0.5 M NaCl pH 3.0 solution. Data sets were analyzed using BiaEval Software. In some cases binding curves could perfectly or not at all be matched with the theoretical curves underlying the evaluation software. In the latter case the binding was determined as “positive”; in the case of closer match numbers are presented but have to be seen as approximation.

[0265] Binding affinities of the respective BiTE antibodies to serum albumin of different species as determined by Biacore are summarized in table 5.

TABLE-US-00005 TABLE 5 Affinities (KD) of tagged PSMA BiTE antibody against serum albumins of different species measured in Biacore experiments (surface plasmon resonance). KD [nM] KD [nM] KD [nM] KD [nM] BiTE antibody HSA RSA MSA BSA PSMA × SA04 negative positive positive negative PSMA × SA08 116 42 77 434 PSMA × SA21 137 49 70 positive PSMA × SA25 1650 95 147  positive PSMA × DX236 3060 negative negative negative PSMA × DX321 positive positive negative negative PSMA × AB01 17400 810  negative negative PSMA × AB14 282 94 negative negative PSMA × AB156 426 96 negative negative HSA = human serum albumin, RSA = rhesus serum albumin, MSA = mouse serum albumin, BSA = bovine serum albumin. “Positive” means detectable binding which could not be determined accurately by the BiaEval software; “Negative” means no significant binding under the chosen experimental conditions.

[0266] As depicted in table 5, all of the tested PSMA BiTE antibodies containing different serum binding peptide tags showed significant binding to human serum albumin with the exception of PSMA×SA04 that were negative on human serum albumin but showed distinct binding to rhesus serum albumin. Moreover, all of the tagged BiTE antibodies tested showed distinct binding to rhesus serum albumin, except for PSMA-DX236. Furthermore, of the tested BiTE antibodies, PSMA×SA04, PSMA×SA08, PSMA×SA21 and PSMA×SA25 showed distinct binding to mouse serum albumin, whereas PSMA×DX236, PSMA×DX321, PSMA×AB1, PSMA×AB14 and PSMA×AB156 not show significant binding to murine serum albumin.

[0267] Among the molecules tested, only PSMA×SA08, PSMA×SA21 and PSMA×SA25 showed distinct binding to bovine serum albumin.

Example 4: Purification Scheme of BiTE Antibodies with/without a C-Terminal ABP

[0268] Chinese hamster ovary CHO cells stable transfected with a pEF DHFR vector coding the BiTE antibody sequence were cultivated in roller bottles till 60% remaining vitality was reached. Cell culture supernatant was cleared by 0.2 μm filtration and applied to a column filled with a zinc loaded chelating gel Fractogel EMD Chelate (Merck, Darmstadt) for immobilized metal affinity chromatography (IMAC) capture.

[0269] After sample loading remaining cell culture supernatant was washed out and after a pre-elution step the HIS6 tagged BiTE antibodies were eluted by applying a buffer containing 500 mM imidazol.

[0270] Eluted volume containing the BiTE antibodies was given on a Superdex S200 (GE Healthcare, Munich) size exclusion chromatography (SEC) column to separate the BiTE monomeric protein from dimeric BiTE protein and other proteins.

[0271] Fractions containing the BiTE monomeric protein were pooled and protein concentration was determined by measurement of optical absorption at 280 nm wavelength and 1 cm lightpath length and multiplication with the protein sequence specific absorption factor.

[0272] BiTE Monomer percentage was calculated by the area under the curve in the SEC chromatograms of the BiTE dimer and BiTE monomer fraction. Monomer percentage=(Area of Monomer/(Area Monomer+Area Dimer))*100.

[0273] As apparent from the tables below (tables 6 to 8) for all tested BiTE antibody variants of anti PSMA BiTE and anti human EpCAM BiTE favorable yield of BiTE monomeric protein and monomer percentage were achieved.

TABLE-US-00006 TABLE 6 BiTE antibody monomer yield and monomer percentage of anti PSMA BiTE variants equipped with a C-terminal added albumin binding peptide ABP showing favorable yield and monomer percentage above 80% Monomer Yield BiTE Antibody BiTE Antibody [μg/L supernatant] Monomer % PSMA × CD3 × SA04 3070 90 PSMA × CD3 × SA08 2243 85 PSMA × CD3 × SA21 1537 84 PSMA × CD3 × SA25 3384 85 PSMA × CD3 × AB01 2572 92 PSMA × CD3 × AB14 4337 88 PSMA × CD3 × AB156 2605 93 PSMA × CD3 × DX236 716 87 PSMA × CD3 × DX321 13392 85

TABLE-US-00007 TABLE 7 Results of the Flow cytometry screening of samples of cell culture supernatants of anti PSMA BiTE variants, equipped with a n-terminal added albumin binding peptide ABP for binding to cells expressing the PSMA antigen prior to purification. All cell culture supernatants not showing any binding in this assay were excluded from the purification procedure. Monomer BiTE Antibody FACS Binding Percentage SA04 × PSMA × CD3 No functional protein (FACS) — SA08 × PSMA × CD3 No functional protein (FACS) — SA21 × PSMA × CD3 No functional protein (FACS) — SA25 × PSMA × CD3 No functional protein (FACS) — AB01 × PSMA × CD3 No functional protein (FACS) — AB14 × PSMA × CD3 No functional protein (FACS) — AB156 × PSMA × CD3 No functional protein (FACS) — DX232 × PSMA × CD3 No functional protein (FACS) — DX236 × PSMA × CD3 No functional protein (FACS) — DX321 × PSMA × CD3 No functional protein (FACS) —

TABLE-US-00008 TABLE 8 BiTE antibody monomer yield and monomer percentage of anti human EpCAM BiTE variant equipped with a C-terminal added albumin binding peptide ABP showing favorable yield and monomer percentage above 80% Monomer Yield BiTE Antibody BiTE molecule [μg/L supernatant] Monomer % EpCAM × CD3 × SA21 4390 91

Example 5: Human EpCAM BiTE Molecules

[0274] Examples for parental human EpCAM BiTE molecules are described in WO 2005/040220.

[0275] Bioactivity of EpCAM BiTE was assessed in a cell-based assay making use of the Toxilight BioAssay Kit (Cambrex/Lonza). This Kit allows measuring the cytotoxic effect of human PBMCs (effector cells) on the EpCAM-expressing breast cancer cell line MDA-MB-453 in presence of EpCAM BITE, by quantification of dead cells according to the following principle:

[0276] The assay was based on the bioluminescent measurement of the enzyme adenylate kinase (AK). AK was present in all cells. Loss of cell membrane integrity by dead cells results in the leakage of AK in the surrounding medium. Addition of the ToxiLight reaction reagent (containing ADP and the enzyme luciferase) triggers a two-step reaction resulting in light emission, which can be measured using a luminometer. The emitted light is linearly related to the AK concentration and correlates with the number of dead cells.

##STR00001##

[0277] Performance and Data Analysis:

[0278] Effector (human PBMC, 2×10.sup.6/well) and target cells (MDA-MB-453, 1×10.sup.5/well) are co-cultivated in an effector to target ratio of 20:1 in presence of different EpCAM BITE concentrations (250 ng/mL-4.2 pg/mL). After 16 to 20 hours the ToxiLight Reagent is added and luminescence is measured (for detailed description see SOP-BIA-110-012).

[0279] The % specific lysis is calculated for each EpCAM BiTE concentration and the data are fitted for determination of half-maximal cytotoxicity (EC.sub.50). Although the EC.sub.50 value describes the biological potency of EpCAM BiTE, the absolute values can vary which is normal for a cell-based assay with different effector cell donors. Thus a relative potency is calculated for each dose response analysis in comparison to a EpCAM BiTE working standard based on the following formula:

Relative Potency=EC.sub.50 sample/EC.sub.50 standard

[0280] Validity of the relative potency determination is controlled by various parameters e.g. curve fit, ratio of curve amplitudes, ratio of slopes, etc.

[0281] The initial cytotoxicity assay conditions do not contain HSA as the assay is developed to test clinical trial material for release and stability.

[0282] Addition of human serum was tested and relative bioactivity of the EpCAM BiTE-SA21 construct was 51-66% for the 10% serum condition and 59-66% for the 20% serum condition (table 13). So the reduction in bioactivity by covalently adding the HSA-binding peptide and using the conditions described was in the range of 50-60%.

[0283] A more pronounced reduction in relative bioactivity was observed for the EpCAM BiTE construct that contained the covalently linked HSA. But also here the bioactivity was not completely blocked; remaining bioactivities were 19-31% and 24-29% for 10 and 20% human serum conditions (table 14).

[0284] Different assay conditions, like addition of FCS or HSA did not alter the observed effect on the relative bioactivity (table 9)

[0285] Dose response curves of the experiments described are shown in FIGS. 3 and 4.

TABLE-US-00009 TABLE 9 EC.sub.50 values for EpCAM BiTE variants EC.sub.50 [pM] EpCAM BiTE -SA21 -HSA with FCS  25 22 176 w/o FCS (864) 602 2147 10% hu Serum  69 105 358 20% hu Serum  99 169 406 8 g/l HSA 428 409 566 20 g/l HSA 128 367 567 10% hu Serum 217 425 700 20% hu Serum 263 400 918

TABLE-US-00010 TABLE 10 Relative bioactivity compared to unmodified EpCAM BiTE EpCAM BiTE-SA21 10% hu Serum 51% 20% hu Serum 66% 10% hu Serum 66% 20% hu Serum 59%

TABLE-US-00011 TABLE 11 Relative bioactivity compared to unmodified EpCAM BiTE EpCAM BiTE-HSA 10% hu Serum 19% 20% hu Serum 24% EpCAM BiTE-HSA- 10% hu Serum 31% 20% hu Serum 29%

Example 7: Generation of Stably Transfected HIV Gp140 Expressing CHO Cells

[0286] The extracellular domain of HIV gp160 (gp140) of the HIV-1 HXB2 strain (Uniprot KB, Ace. No. P04578) was fused to the transmembrane and intracellular domain of EpCAM. The open reading frame of this fusion protein was cloned into a pEF DHFR vector according to standard procedures (the general procedure for the transfection of CHO cells with cell surface molecules to create appropriate target cells is derivable from WO 2008/119567) and then used for the analyses of the designated bispecific constructs.

Example 8: Generation of HIV Bispecific Constructs

[0287] The HIV target binding domain of the bispecific constructs described below are of human CD4 origin. Constructs named CD4(1+2) contain the first two domains of human CD4. Constructs CD4 D1.1 or CD4 D1.2 are of human CD4 domain 1 origin containing single point mutations and are described by Chen, W., et al. (2011, J Virol 85(18): 9395-9405). The two constructs described here represent the proof of their applicability in the BiTE context, but is not limited to these two CD4 domain 1 constructs i.e. the described bispecific format can be applied to all described variants thereof, especially concerning the published variants CD4 D1.3 to CD4 D1.19. Generally, molecules with intrinsic binding moieties to HIV proteins accessible at the plasma membrane are applicable in the bispecific context, e.g. constructs containing only parts of human CD4 like the core peptide sequence, aptamers of the human CD4 protein or chimeras of e.g. rat CD4 with human CD4 insertions involved in gp120 binding as illustrated by James H. M. Simon (1993, J. Exp. Med, Volume 177, April 1993, 949-954) can be used in the BiTE context. Next to binding domains of human CD4 origin, antibody specificities of anti HIV human monoclonal antibodies such as B12, VRC01 or 4E10 in scFv format in VH-VL or VL-VH orientation are also applicable in the BiTE context and have been characterized concerning their bispecific binding and cytotoxic activity. The described HIV gp120 binding domains were fused to a cross-specific human CD3 binder. Long lived version of these parental HIV specific constructs have been designed and cloned according to standard procedures and are characterized below.

Example 9: Cytotoxic Activity Bispecific Constructs for HIV Positive Target Cells

[0288] FACS Based Cytotoxicity Assay with Stimulated Human T-Cells:

Stimulated T-cells enriched for CD8.sup.+ T-cells were obtained as described below.
A petri dish (145 mm diameter, Greiner bio-one GmbH, Kremsmünster) was coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) and anti-CD28 (Art. No. 340975, BD) in a final concentration of 1 μg/ml for 1 hour at 37° C. Unbound protein was removed by one washing step with PBS. 3-5×10.sup.7 human PBMC were added to the precoated petri dish in 100 ml of RPMI 1640 with stabilized glutamine/10% FCS/IL-2 20 U/ml (Proleukin®, Chiron) and stimulated for 3 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 cell culture medium mentioned above.

[0289] CD8.sup.+ cytotoxic T lymphocytes (CTLs) were enriched by depletion of non CD8.sup.+ cells using a human CD8.sup.+ T Cell Isolation Kit according to the manufacturer's (Miltenyi Biotec) protocol.

[0290] HIV gp140-transfected CHO target cells (the general procedure for the transfection of CHO cells with cell surface molecules to create appropriate target cells is derivable from WO 2008/119567) were washed with PBS plus 2% FCS and labeled with Vybrant® DiD cell-labeling solution (Invitrogen, 125,000 cells/ml, 5 μl dye per 10.sup.6 cells) in PBS plus 2% FCS for 3 minutes at 37° C. Subsequently, the labeled target cells were washed 3 times with 50 ml PBS plus 2% FCS and then used in the cytotoxicity assay. The assay was performed in a 96-well plate in a total volume of 200 μl supplemented RPMI medium (as above) with an Effector:Target ratio of 10:1. A starting concentration of 2-10 μg/ml of purified bispecific antibody and three to four-fold dilutions thereof were used. Incubation time for the assay was 18-24 hours.

[0291] After the incubation, the cells were resuspended and transferred into a 96 well v-bottom plate. Adhering target cells were trypsinized for 5 minutes at 37° C., resuspended in PBS/2% FCS and combined with the other cells from the respective wells. The plates were centrifuged for 4 minutes at 1200 rpm, the supernatant was discarded and the cells were resuspended in PBS plus 2% FCS plus 1 μg/ml propidium iodide (PI) (supplied by Sigma Aldrich). Cytotoxicity was determined as percentage of DiD/PI double positive cells relative to the overall number of DiD positive cells as determined by flow cytometry using a FACSCanto II (BD Biosciences, Heidelberg, Germany). All measurements were carried out in duplicates. Analyses of the results were carried out with Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, Calif., USA). EC.sub.50 values were calculated by the analysis program from the sigmoidal dose or variable slope response curves and then used for comparison of cytotoxic activity.

[0292] The results for the cytotoxic activity using RPMI 1640 supplemented with 10% FCS or 50% Human Serum (PAA Laboratories GmbH, Art. No. C11-021) respectively on HIV gp140 expressing target cells are shown in FIG. 5(1)-5(4).

[0293] EC.sub.50 values (i.e. BiTE concentration at half maximal lysis of target cells) are shown for the designated bispecific molecules in tables 12 to 17 to illustrate their cytotoxic activity.

TABLE-US-00012 TABLE 12 EC.sub.50 values of N-terminal ABP tagged BiTE antibodies under the influence of 10% FCS BiTE CD4(1 + 2)xaCD3 SA21xCD4(1 + 2)xaCD3 EC.sub.50 [ng/ml] 0.12 0.93 Factor* 1 7.75 BiTE SA21LxCD4(1 + 2)xaCD3 SA21LxCD4(1 + 2)xaCD3 EC.sub.50 [ng/ml] 2.0 0.78 Factor* 16.6 6.5 BiTE SA25xCD4(1 + 2)xaCD3 EC.sub.50 [ng/ml] 0.29 Factor* 2.41 Factor*: Factor describes the EC.sub.50 value difference of the designated bispecific molecule compared to the parental bispecific molecule without ABP tag.

TABLE-US-00013 TABLE 13 EC.sub.50 values of N-terminal ABP tagged BiTE antibodies under the influence of 50% Human Serum BiTE CD4(1 + 2)xaCD3 SA21xCD4(1 + 2)xaCD3 EC.sub.50 [ng/ml] 0.24 4.0 Factor* 1 16.6 BiTE SA21LxCD4(1 + 2)xaCD3 SA21LxCD4(1 + 2)xaCD3 EC.sub.50 [ng/ml] 5.5 2.7 Factor* 22.9 11.25 BiTE SAx25CD4(1 + 2)xaCD3 EC.sub.50 [ng/ml] 0.78 Factor* 3.25 Factor*: Factor describes the EC.sub.50 value difference of the designated bispecific molecule compared to the parental bispecific molecule without ABP tag.

TABLE-US-00014 TABLE 14 EC.sub.50 values of C-terminal ABP tagged BiTE antibodies under the influence of 10% FCS BiTE CD4(1 + 2)xaCD3 CD4(1 + 2)xaCD3xSA21 EC.sub.50 [ng/ml] 0.12 0.23 Factor* 1   1.92 BiTE CD4(1 + 2)xaCD3LxSA21 CD4(1 + 2)xaCD3LxSA21 EC.sub.50 [ng/ml] 0.23 0.35 Factor* 1.92 2.92 BiTE CD4(1 + 2)xaCD3xSA25 EC.sub.50 [ng/ml] 0.21 Factor* 1.75 Factor*: Factor describes the EC.sub.50 value difference of the designated bispecific molecule compared to the parental bispecific molecule without ABP tag.

TABLE-US-00015 TABLE 15 EC.sub.50 values of C-terminal ABP tagged BiTE antibodies under the influence of 50% Human Serum BiTE CD4(1 + 2)xaCD3 CD4(1 + 2)xaCD3xSA21 EC.sub.50 [ng/ml] 0.24 1.2 Factor* 1 5 BiTE CD4(1 + 2)xaCD3LxSA21 CD4(1 + 2)xaCD3LxSA21 EC.sub.50 [ng/ml] 1.2 1.7 Factor* 5 7.08 BiTE CD4(1 + 2)xaCD3xSA25 EC.sub.50 [ng/ml] 0.74 Factor* 3.08 Factor*: Factor describes the EC.sub.50 value difference of the designated bispecific molecule compared to the parental bispecific molecule without ABP tag.

[0294] Comparing the EC.sub.50 values of C-terminal SA21 tagged bispecific molecules (Table 15, FIG. 5(4)) with the N-terminal SA21 tagged bispecific molecules (Table 13, FIG. 5(2)) indicates a significant negative influence in the cytotoxic activity for N-terminal SA21 tagged bispecific molecules indicated by increased EC.sub.50 values in presence of 50% Human Serum. This phenomenon can be observed irrelevant of the position of the SA21 tag at the molecule's terminus, i.e. no restoration of cytotoxic activity by the addition of five AA or 15 AA linker between the ABP tag and the neighboring binding domain. In numbers, N-terminal SA21 tagged BiTE molecules show a 11 to 23 fold decrease in cytotoxic activity compared to 5 to 7 fold decrease when the SA21 tag is added to the C-terminus of the bispecific molecule. The described observation can also be seen in the presence of 10% FCS, i.e. less influence of serum albumin due to lower number of molecules and different species. A decrease of six to 16 fold for N-terminal SA21 tagged bispecific molecules (Table 12) compared to 2 to 3 fold decrease for C-terminal SA21 tagged BiTE antibodies (Table 14).

TABLE-US-00016 TABLE 16 EC.sub.50 values of BiTE antibodies ABP tagged between the target and CD3 binding domain under the influence of 10% FCS BiTE CD4(1 + 2)xaCD3 CD4(1 + 2)SA21xaCD3 EC.sub.50 0.055 0.75 [ng/ml] Factor* 1 13.64 BiTE CD4(1 + 2)LxSA21LxaCD3 CD4(1 + 2)LxSA21LxaCD3 EC.sub.50 0.99 3.7 [ng/ml] Factor* 18 67.28 Factor*: Factor describes the EC.sub.50 value difference of the designated bispecific molecule compared to the parental bispecific molecule without ABP tag.

TABLE-US-00017 TABLE 17 EC.sub.50 values of BiTE antibodies ABP tagged between the target and CD3 binding domain under the influence of 50% Human Serum BiTE CD4(1 + 2)xaCD3 CD4(1 + 2)SA21xaCD3 EC.sub.50 [ng/ml] 0.11 5.1 Factor* 1 43.36 BiTE CD4(1 + 2)LSA21LxaCD3 CD4(1 + 2)LxSA21LxaCD3 EC.sub.50 [ng/ml] 2.9 ~8.0 Factor* 26.36 72.72 Factor*: Factor describes the EC.sub.50 value difference of the designated bispecific molecule compared to the parental bispecific molecule without ABP tag.

[0295] Locating the SA21 ABP tag between the HIV and CD3 binding specificities leads to a decrease in cytotoxic activity in the presence of 10% FCS dependent of the length of the ABP tag insertion (Table 16 and FIG. 5(5)). This decrease in cytotoxic activity is partially exaggerated in the presence of 50% Human Serum (Table 17 and FIG. 5(6)).

Example 10: Biacore-Based Determination of BiTE-Antibody Affinity to Serum Albumin of Different Species

[0296] To confirm binding of the tagged HIV specific BiTE molecules to human serum albumin and serum albumins of other species, the following serum albumin preparations were provided: Human serum albumin (HSA), Sigma A9511; Mouse serum albumin (MSA), Sigma A3139;

[0297] Binding experiments were performed using plasmon resonance measurements in a Biacore machine. In detail, CM5 Sensor Chips (GE Healthcare) were immobilized with the respective serum albumin protein using acetate buffer pH 3 according to the manufacturer's manual. The BiTE antibody samples were loaded five relevant concentrations e.g.: 400 nM, 200 nM, 100 nM, 50 nM, 25 nM (low concentrated BiTE's concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM) diluted in HBS-EP running buffer (GE Healthcare). For affinity determinations the flow rate was 35 μl/min for 3 min, then HBS-EP running buffer was applied for 6 min again at a flow rate of 35 μl/ml. Regeneration of the chip was performed using a buffer consisting of 100 mM glycine 0.5 M NaCl pH 3.0 solution for 60 seconds. Data sets were analyzed using BiaEval Software. In some cases binding curves could perfectly or not at all be matched with the theoretical curves underlying the evaluation software. In the latter case the binding was determined as “positive”; in the case of closer match numbers are presented but have to be seen as approximation.

[0298] Binding affinities of the respective BiTE antibodies to HSA as determined by Biacore are summarized in table 18 and FIG. 6.

TABLE-US-00018 TABLE 18 Affinities (KD) of tagged HIV BiTE antibody against human serum albumin measured in Biacore experiments (surface plasmon resonance). KD [nM] KD [nM] BiTE antibody HSA MSA CD4(1 + 2)xaCD3 Negative Negative SA21CD4(1 + 2)xaCD3 226 87 SA21LCD4(1 + 2)xaCD3 536 72 SA21LxCD4(1 + 2)xaCD3 260 107  SA25CD4(1 + 2)xaCD3 Positive Positive CD4(1 + 2)xaCD3SA21 201 113  CD4(1 + 2)xaCD3LSA21 Positive Positive CD4(1 + 2)xaCD3LxSA21 Positive Positive CD4(1 + 2)xaCD3SA25 198 63 CD4(1 + 2)SA21aCD3 465 70 CD4(1 + 2)LSA21LaCD3 773 231  CD4(1 + 2)LxSA21LxI2C 4520  8210  HSA = human serum albumine, MSA = mouse serum albumin, “Positive” means detectable binding which could not be determined accurately by the BiaEval software; “Negative” means no significant binding under the chosen experimental conditions.

[0299] Concerning the affinity of the ABP tagged bispecific molecules they all bound to the immobilized HSA and MSA proteins. The bispecific molecules harboring the ABP tag between their binding specificities showed worse affinities to HSA and MSA than the bispecific molecules tagged with SA21 or SA25 at either one of their termini.

Example 11: Biacore-Based Determination of BiTE-Antibody Affinity to Human CD3

[0300] To confirm binding of the C-terminal ABP tagged HIV BiTE molecules to human CD3, a human CD3 peptide comprising the CD3 epitope was fused to human Fc according to standard procedures and used for the affinity measurement.

[0301] Binding experiments were performed using plasmon resonance measurements in a Biacore machine. In detail, CM5 Sensor Chips (GE Healthcare) were immobilized with the human CD3×huFc protein using acetate buffer pH 4.5 according to the manufacturer's manual. The BiTE antibody samples were loaded five relevant concentrations e.g.: 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM diluted in HBS-EP running buffer (GE Healthcare). For affinity determinations the flow rate was 35 μl/min for 3 min, then HBS-EP running buffer was applied for 8 min again at a flow rate of 35 μl/ml. Regeneration of the chip was performed using a buffer consisting of 100 mM glycine 0.5 M NaCl pH 1.5 solution for 60 seconds. Data sets were analyzed using BiaEval Software.

TABLE-US-00019 TABLE 19 Affinities (KD) of tagged HIV BiTE antibody against human CD3 FC fusion protein measured in Biacore experiments (surface plasmon resonance). KD [nM] BiTE antibody huCD3 Factor* CD4(1 + 2)xaCD3 2.31 1 CD4(1 + 2)xaCD3xSA21 4.33 1.9 CD4(1 + 2)xaCD3xSA25 3.77 1.6 Factor*: Decreased CD3 affinity in comparison to parental BiTE molecule without ABP tag

[0302] The C-terminal addition of an ABP tag leads to a maximal 2 fold decrease of the neighboring binding specificity (i.e. CD3), see table 19 and FIG. 7.

Example 12: Biacore-Based Determination of BiTE-Antibody Affinity to HIV Gp120

[0303] To confirm binding of the N-terminal ABP tagged HIV BiTE molecules to HIV gp120, the following HIV gp120 was provided: Recombinant HIV-1 IIIB Envelope Glycoprotein gp120 (Baculovirus), Product #1001, Immunodiagnostics, Incorporation. Binding experiments were performed using plasmon resonance measurements in a Biacore machine. In detail, CM5 Sensor Chips (GE Healthcare) were immobilized with the respective HIV gp120 protein using acetate buffer pH 4.5 according to the manufacturer's manual. The BiTE antibody samples were loaded five relevant concentrations e.g.: 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM diluted in HBS-EP running buffer (GE Healthcare). For affinity determinations the flow rate was 35 μl/min for 3 min, then HBS-EP running buffer was applied for 7 min again at a flow rate of 35 μl/ml. Regeneration of the chip was performed using a buffer consisting of 4 M MgCl2 solution for 60 seconds. Data sets were analyzed using BiaEval Software.

TABLE-US-00020 TABLE 20 Affinities (KD) of tagged HIV BiTE antibody against HIV gp120 protein measured in Biacore experiments (surface plasmon resonance). KD [nM] BiTE antibody gp120 Factor* CD4(1 + 2)xaCD3 0.71 1 SA21CD4(1 + 2)xaCD3 4.61 6.5 SA25CD4(1 + 2)xaCD3 10.3 14.5 Factor*: Decreased HIV gp120 affinity in comparison to parental BiTE molecule without ABP tag

[0304] The N-terminal addition of an ABP tag leads to 6 to 14 fold decrease of the neighboring binding specificity (i.e. anti HIV gp120), see table 20 and FIG. 8.

Example 13: Purification Scheme of BiTE Antibodies with a c-Terminal ABP

[0305] Human embryonal kidney (HEK 293-F, Invitrogen) cells were transiently transfected with 200 μg of a pEF DHFR vector coding the BiTE antibody sequence using 293 fectin (according to the manufacturer's manual, Invitrogen). Transfected cells were cultivated in Erlenmeyer cell culture flasks for 72 hours at 37° C., 135 rpm, 5% CO.sub.2. Alternatively, Chinese hamster ovary CHO cells stably transfected with a pEF DHFR vector coding the BiTE antibody sequence were cultivated in roller bottles till 60% remaining vitality was reached. Cell culture supernatant was cleared by centrifugation at 4′000 rpm for 10 minutes and subsequent filtration (0.2 μm) and applied to a column filled with a zinc loaded chelating gel Fractogel EMD Chelate (Merck, Darmstadt) for immobilized metal affinity chromatography (IMAC) capture.

[0306] After sample loading remaining cell culture supernatant was washed out and after a pre-elution step the HIS6 tagged BiTE antibodies were eluted by applying a buffer containing 500 mM imidazol.

[0307] Eluted volume containing the BiTE antibodies was given on a Superdex S200 (GE Healthcare, Munich) size exclusion chromatography (SEC) column to separate the BiTE monomeric protein from dimeric BiTE protein and other proteins.

[0308] Fractions containing the BiTE monomeric protein were pooled and protein concentration was determined by measurement of optical absorption at 280 nm wavelength and 1 cm lightpath length and multiplication with the protein sequence specific absorption factor.

[0309] Exemplary purification showing the SEC chromatogram of the unmodified CD4(1+2)×aCD3L×SA21 BiTE is shown in FIG. 9.

[0310] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0311] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0312] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0313] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

TABLE-US-00021 Sequences SEQ ID NO. DESIGNATION SOURCE TYPE SEQUENCE 1 Human human aa QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEI CD3ϵ LWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQS extracellular GYYVCYPRGSKPEDANFYLYLRARVCENCMEMD domain 2 Human CD3ϵ 1- human aa QDGNEEMGGITQTPYKVSISGTTVILT 27 3 Callithrix Callithrix aa QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEI jacchus CD3ϵ jacchus KWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKE extracellular TPAEEASHYLYLKARVCENCVEVD domain 4 Callithrix Callithrix aa QDGNEEMGDTTQNPYKVSISGTTVTLT jacchus CD3ϵ 1- jacchus 27 5 Saguinus Saguinus aa QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEI oedipus CD3ϵ oedipus KWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKE extracellular TPAEEASHYLYLKARVCENCVEVD domain 6 Saguinus Saguinus aa QDGNEEMGDTTQNPYKVSISGTTVTLT oedipus CD3ϵ 1- oedipus 27 7 Saimiri Saimiri aa QDGNEEIGDTTQNPYKVSISGTTVTLTCPRYDGQEI sciureus sciureus KWLVNDQNKEGHEDHLLLEDFSEMEQSGYYACLSKE CD3ϵ TPTEEASHYLYLKARVCENCVEVD extracellular domain 8 Saimiri Saimiri aa QDGNEEIGDTTQNPYKVSISGTTVTLT sciureus sciureus CD3ϵ 1-27 9 SA04 artificial aa DICLPRWGCLW 10 SA04 artificial nt GACATCTGCCTGCCTAGATGGGGCTGCCTGTGG 11 SA08 artificial aa QGLIGDICLPRWGCLWGDSVK 12 SA08 artificial nt CGGCTGATCGAGGACATCTGCCTGCCCAGATGGGGC TGCCTGTGGGAGGACGAC 13 SA21 artificial aa RLIEDICLPRWGCLWEDD 14 SA21 artificial nt CGGCTGATCGAGGACATCTGCCTGCCCAGATGGGGC TGCCTGTGGGAGGACGAC 15 SA25 artificial aa EDICLPRWGCLWED 16 SA25 artificial nt GAGGACATCTGCCTGCCCAGATGGGGCTGCCTGTGG GAGGAC 17 DX236 artificial aa AEGTGDFWFCDRIAWYPQHLCEFLDPE 18 DX236 artificial nt GCTGAGGGCACCGGCGATTTCTGGTTCTGCGACCGG ATCGCCTGGTATCCCCAGCACCTGTGCGAGTTTCTG GACCCCGAA 19 DX321 artificial aa AEGTGDRNMCKFSWIRSPAFCARADPE 20 DX321 artificial nt GCTGAGGGCACCGGCGACCGGAACATGTGCAAGTTC AGCTGGATCCGGTCCCCCGCCTTCTGCGCCAGAGCC GACCCTGAA 21 AB01 artificial aa AASYSDYDVFGGGTDFGP 22 AB01 artificial nt GCCGCTAGCTACTCCGACTACGACGTGTTCGGCGGA GGCACCGACTTCGGGCCA 23 AB14 artificial aa AARYWDYDVFGGGTPVGG 24 AB14 artificial nt GCCGCTCGGTACTGGGACTACGACGTGTTCGGCGGA GGCACACCCGTGGGGGGC 25 AB156 artificial aa AARDWDFDVFGGGTPVGG 26 AB156 artificial nt GCCGCTCGGGACTGGGACTTCGACGTGTTCGGCGGA GGCACACCCGTGGGGGGC 27 EpCAM × CD3 × artificial nt GAGCTCGTGATGACACAGTCTCCATCCTCCCTGACT SA21 × His-tag GTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAG TCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAG AACTACTTGACCTGGTACCAGCAGAAACCAGGGCAG CCTCCTAAACTGTTGATCTACTGGGCATCCACTAGG GAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGA TCTGGAACAGATTTCACTCTCACCATCAGCAGTGTG CAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAAT GATTATAGTTATCCGCTCACGTTCGGTGCTGGGACC AAGCTTGAGATCAAAGGTGGTGGTGGTTCTGGCGGC GGCGGCTCCGGTGGTGGTGGTTCTGAGGTGCAGCTG CTCGAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGG ACTTCAGTGAAGATATCCTGCAAGGCTTCTGGATAC GCCTTCACTAACTACTGGCTAGGTTGGGTAAAGCAG AGGCCTGGACATGGACTTGAGTGGATTGGAGATATT TTCCCTGGAAGTGGTAATATCCACTACAATGAGAAG TTCAAGGGCAAAGCCACACTGACTGCAGACAAATCT TCGAGCACAGCCTATATGCAGCTCAGTAGCCTGACA TTTGAGGACTCTGCTGTCTATTTCTGTGCAAGACTG AGGAACTGGGACGAGCCTATGGACTACTGGGGCCAA GGGACCACGGTCACCGTCTCCTCCGGAGGTGGTGGC TCCGACGTCCAACTGGTGCAGTCAGGGGCTGAAGTG AAAAAACCTGGGGCCTCAGTGAAGGTGTCCTGCAAG GCTTCTGGCTACACCTTTACTAGGTACACGATGCAC TGGGTAAGGCAGGCACCTGGACAGGGTCTGGAATGG ATTGGATACATTAATCCTAGCCGTGGTTATACTAAT TACGCAGACAGCGTCAAGGGCCGCTTCACAATCACT ACAGACAAATCCACCAGCACAGCCTACATGGAACTG AGCAGCCTGCGTTCTGAGGACACTGCAACCTATTAC TGTGCAAGATATTATGATGATCATTACTGCCTTGAC TACTGGGGCCAAGGCACCACGGTCACCGTCTCCTCA GGCGAAGGTACTAGTACTGGTTCTGGTGGAAGTGGA GGTTCAGGTGGAGCAGACGACATTGTACTGACCCAG TCTCCAGCAACTCTGTCTCTGTCTCCAGGGGAGCGT GCCACCCTGAGCTGCAGAGCCAGTCAAAGTGTAAGT TACATGAACTGGTACCAGCAGAAGCCGGGCAAGGCA CCCAAAAGATGGATTTATGACACATCCAAAGTGGCT TCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCT GGGACCGACTACTCTCTCACAATCAACAGCTTGGAG GCTGAAGATGCTGCCACTTATTACTGCCAACAGTGG AGTAGTAACCCGCTCACGTTCGGTGGCGGGACCAAG GTGGAGATCAAACGGCTGATCGAGGACATCTGCCTG CCCAGATGGGGCTGCCTGTGGGAGGACGAC CATCATCACCATCATCAT 28 EpCAM × CD3 × artificial aa ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQK SA21 × His-tag NYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSG SGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGT KLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPG TSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDI FPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLT FEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGG SDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMH WVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTIT TDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLD YWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQ SPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKA PKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLE AEDAATYYCQQWSSNPLTFGGGTKVEIKRLIEDICL PRWGCLWEDDHHHHHH 29 EpCAM × CD3 × artificial aa ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQK SA21 NYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSG SGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGT KLEIKGGGGSGGGGSGGGGSEVQLLEQSGAELVRPG TSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDI FPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLT FEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSGGGG SDVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMH WVRQAPGQGLEWIGYINPSRGYTNYADSVKGRFTIT TDKSTSTAYMELSSLRSEDTATYYCARYYDDHYCLD YWGQGTTVTVSSGEGTSTGSGGSGGSGGADDIVLTQ SPATLSLSPGERATLSCRASQSVSYMNWYQQKPGKA PKRWIYDTSKVASGVPARFSGSGSGTDYSLTINSLE AEDAATYYCQQWSSNPLTFGGGTKVEIKRLIEDICL PRWGCLWEDD 30 CD4(1 + 2) artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCCAGACGGTCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACAGCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACCCTGGAAAGC CCCCCTGGCTCCAGCCCTTCCGTGCAGTGCCGGTCC CCTCGGGGCAAGAACATCCAGGGCGGCAAGACCCTG TCCGTGTCCCAGCTGGAACTGCAGGACAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAATTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAGGCT 31 CD4(1 + 2) artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KA 32 CD4(1 + 2) × aCD3 artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG × His tag GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCCAGACGGTCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACAGCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACCCTGGAAAGC CCCCCTGGCTCCAGCCCTTCCGTGCAGTGCCGGTCC CCTCGGGGCAAGAACATCCAGGGCGGCAAGACCCTG TCCGTGTCCCAGCTGGAACTGCAGGACAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAATTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAGGCTTCCGGAGGTGGTGGATCCGAGGTGCAGCTG GTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGG TCATTGAAACTCTCATGTGCAGCCTCTGGATTCACC TTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCT CCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGA AGTAAATATAATAATTATGCAACATATTATGCCGAT TCAGTGAAAGACAGGTTCACCATCTCCAGAGATGAT TCAAAAAACACTGCCTATCTACAAATGAACAACTTG AAGACTGAGGACACTGCCGTGTACTACTGTGTGAGA CATGGGAACTTCGGTAATAGCTACATATCCTACTGG GCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCC TCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGT GGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCT TCACTCACCGTATCACCTGGTGGAACAGTCACACTC ACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGC AACTACCCAAACTGGGTCCAACAAAAACCAGGTCAG GCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTC GCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTG CTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTA CAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTA TGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACC AAACTGACTGTCCTACATCATCACCATCATCAT 33 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI × His tag LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLHHHHHH 34 CD4 D1.1 artificial NT AAGAAAGTCGTGATCGGCAAGAAAGGCGACACCGTG GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGTGGACTCTCGGAGATCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGCCCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGATC GTGCTGGGC 35 CD4 D1.1 artificial AA KKVVIGKKGDTVELTCTASQKKSIQFHWKNSNQIKI LGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIK NLKPEDSDTYICEVEDQKEEVQLIVLG 33 CD4 artificial NT AAGAAAGTCGTGATCGGCAAGAAAGGCGACACCGTG D1.1 × aCD3 × GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC His tag CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGTGGACTCTCGGAGATCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGCCCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGATC GTGCTGGGCTCCGGAGGTGGTGGATCCGAGGTGCAG CTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGA GGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTC ACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAG GCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATA AGAAGTAAATATAATAATTATGCAACATATTATGCC GATTCAGTGAAAGACAGGTTCACCATCTCCAGAGAT GATTCAAAAAACACTGCCTATCTACAAATGAACAAC TTGAAGACTGAGGACACTGCCGTGTACTACTGTGTG AGACATGGGAACTTCGGTAATAGCTACATATCCTAC TGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTC TCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCC GGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAA CCTTCACTCACCGTATCACCTGGTGGAACAGTCACA CTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCT GGCAACTACCCAAACTGGGTCCAACAAAAACCAGGT CAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTC CTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCC CTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGG GTACAGCCAGAGGATGAGGCAGAATATTACTGTGTT CTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGA ACCAAACTGACTGTCCTACATCATCACCATCATCAT 37 CD4 artificial AA KKVVIGKKGDTVELTCTASQKKSIQFHWKNSNQIKI D1.1 × aCD3 × LGNQGSFLTKGPSKLNDRVDSRRSLWDQGNFPLIIK His tag NLKPEDSDTYICEVEDQKEEVQLIVLGSGGGGSEVQ LVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQ APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD DSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISY WAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSG VQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH 38 CD4 D1.2 artificial NT AAGAAAGTGGTGTACGGCAAGAAAGGCGACACCGTG GAACTGACCTGCACCGCCTCCCAGAAGAAGAACATC CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCTCGGAGATCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGCCCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGGTG GTCGTGGGC 39 CD4 D1.2 artificial AA KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKI LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKPEDSDTYICEVEDQKEEVQLVVVG 40 CD4 artificial NT AAGAAAGTGGTGTACGGCAAGAAAGGCGACACCGTG D1.2 × aCD3 × GAACTGACCTGCACCGCCTCCCAGAAGAAGAACATC His tag CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCTCGGAGATCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGCCCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGGTG GTCGTGGGCTCCGGAGGTGGTGGATCCGAGGTGCAG CTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGA GGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTC ACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAG GCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATA AGAAGTAAATATAATAATTATGCAACATATTATGCC GATTCAGTGAAAGACAGGTTCACCATCTCCAGAGAT GATTCAAAAAACACTGCCTATCTACAAATGAACAAC TTGAAGACTGAGGACACTGCCGTGTACTACTGTGTG AGACATGGGAACTTCGGTAATAGCTACATATCCTAC TGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTC TCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCC GGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAA CCTTCACTCACCGTATCACCTGGTGGAACAGTCACA CTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCT GGCAACTACCCAAACTGGGTCCAACAAAAACCAGGT CAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTC CTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCC CTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGG GTACAGCCAGAGGATGAGGCAGAATATTACTGTGTT CTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGA ACCAAACTGACTGTCCTACATCATCACCATCATCAT 41 CD4 artificial AA KKVVYGKKGDTVELTCTASQKKNIQFHWKNSNQIKI D1.2 × aCD3 × LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK His tag NLKPEDSDTYICEVEDQKEEVQLVVVGSGGGGSEVQ LVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQ APGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD DSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISY WAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQE PSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSG VQPEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH 42 SA21CD4(1 + 2) artificial NT CGGCTGATCGAGGACATCTGCCTGCCTAGATGGGGC TGCCTGTGGGAGGACGACAAGAAAGTGGTGCTGGGC AAGAAAGGCGACACCGTGGAACTGACCTGCACCGCC TCCCAGAAGAAGTCCATCCAGTTCCACTGGAAGAAC TCCAACCAGATCAAGATCCTGGGCAACCAGGGCAGC TTCCTGACCAAGGGCCCCTCCAAGCTGAACGACCGG GCCGACTCTCGGAGATCCCTGTGGGATCAGGGCAAC TTCCCACTGATCATCAAGAACCTGAAGATCGAGGAT TCCGACACCTACATCTGCGAGGTGGAAGATCAGAAA GAAGAGGTGCAGCTGCTGGTGTTCGGCCTGACCGCC AACTCCGATACCCATCTGCTGCAGGGCCAGTCCCTG ACCCTGACACTGGAATCTCCACCCGGCTCCAGCCCT TCCGTGCAGTGCAGATCTCCCAGAGGCAAGAACATC CAGGGCGGCAAGACCCTGTCCGTGTCCCAGCTGGAA CTGCAGGACTCTGGCACCTGGACCTGTACCGTGCTG CAGAACCAGAAAAAGGTGGAATTCAAGATCGACATC GTGGTGCTGGCCTTCCAGAAGGCC 43 SA21CD4(1 + 2) artificial AA RLIEDICLPRWGCLWEDDKKVVLGKKGDTVELTCTA SQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDR ADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQK EEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSP SVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVL QNQKKVEFKIDIVVLAFQKA 44 SA21CD4 artificial NT CGGCTGATCGAGGACATCTGCCTGCCTAGATGGGGC (1 + 2) × TGCCTGTGGGAGGACGACAAGAAAGTGGTGCTGGGC aCD3 × His tag AAGAAAGGCGACACCGTGGAACTGACCTGCACCGCC TCCCAGAAGAAGTCCATCCAGTTCCACTGGAAGAAC TCCAACCAGATCAAGATCCTGGGCAACCAGGGCAGC TTCCTGACCAAGGGCCCCTCCAAGCTGAACGACCGG GCCGACTCTCGGAGATCCCTGTGGGATCAGGGCAAC TTCCCACTGATCATCAAGAACCTGAAGATCGAGGAT TCCGACACCTACATCTGCGAGGTGGAAGATCAGAAA GAAGAGGTGCAGCTGCTGGTGTTCGGCCTGACCGCC AACTCCGATACCCATCTGCTGCAGGGCCAGTCCCTG ACCCTGACACTGGAATCTCCACCCGGCTCCAGCCCT TCCGTGCAGTGCAGATCTCCCAGAGGCAAGAACATC CAGGGCGGCAAGACCCTGTCCGTGTCCCAGCTGGAA CTGCAGGACTCTGGCACCTGGACCTGTACCGTGCTG CAGAACCAGAAAAAGGTGGAATTCAAGATCGACATC GTGGTGCTGGCCTTCCAGAAGGCCTCCGGAGGTGGT GGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGA TTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGT GCAGCCTCTGGATTCACCTTCAATAAGTACGCCATG AACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAA TGGGTTGCTCGCATAAGAAGTAAATATAATAATTAT GCAACATATTATGCCGATTCAGTGAAAGACAGGTTC ACCATCTCCAGAGATGATTCAAAAAACACTGCCTAT CTACAAATGAACAACTTGAAGACTGAGGACACTGCC GTGTACTACTGTGTGAGACATGGGAACTTCGGTAAT AGCTACATATCCTACTGGGCTTACTGGGGCCAAGGG ACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCT GGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACT GTTGTGACTCAGGAACCTTCACTCACCGTATCACCT GGTGGAACAGTCACACTCACTTGTGGCTCCTCGACT GGGGCTGTTACATCTGGCAACTACCCAAACTGGGTC CAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATA GGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCC AGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCC CTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCA GAATATTACTGTGTTCTATGGTACAGCAACCGCTGG GTGTTCGGTGGAGGAACCAAACTGACTGTCCTACAT CATCACCATCATCAT 45 SA21 CD4(1 + 2) × artificial AA RLIEDICLPRWGCLWEDDKKVVLGKKGDTVELTCTA aCD3 × His tag SQKKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDR ADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQK EEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSP SVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCTVL QNQKKVEFKIDIVVLAFQKASGGGGSEVQLVESGGG LVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLE WVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAY LQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQG TLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSP GGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLI GGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWVFGGGTKLTVLHHHHHH 46 SA25CD4(1 + 2) artificial NT GAGGACATCTGCCTGCCCAGATGGGGCTGCCTGTGG GAGGACAAGAAAGTGGTGCTGGGCAAGAAAGGCGAC ACCGTGGAACTGACCTGCACCGCCTCCCAGAAGAAG TCCATCCAGTTCCACTGGAAGAACTCCAACCAGATC AAGATCCTGGGCAACCAGGGCAGCTTCCTGACCAAG GGCCCCTCCAAGCTGAACGACCGGGCCGACTCTCGG AGATCCCTGTGGGATCAGGGCAACTTCCCACTGATC ATCAAGAACCTGAAGATCGAGGACTCCGACACCTAC ATCTGCGAGGTGGAAGATCAGAAAGAAGAGGTGCAG CTGCTGGTGTTCGGCCTGACCGCCAACAGCGACACC CATCTGCTGCAGGGCCAGTCCCTGACCCTGACACTG GAATCTCCACCCGGCTCCAGCCCTTCCGTGCAGTGC AGATCTCCCAGAGGCAAGAACATCCAGGGCGGCAAG ACCCTGTCCGTGTCCCAGCTGGAACTGCAGGACTCT GGCACCTGGACCTGTACCGTGCTGCAGAACCAGAAA AAGGTGGAATTCAAGATCGACATCGTGGTGCTGGCC TTCCAGAAGGCC 47 SA25CD4(1 + 2) artificial AA EDICLPRWGCLWEDKKVVLGKKGDTVELTCTASQKK SIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSR RSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQ LLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQC RSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQK KVEFKIDIVVLAFQKA 48 SA25CD4(1 + 2) × artificial NT GAGGACATCTGCCTGCCCAGATGGGGCTGCCTGTGG aCD3 × His tag GAGGACAAGAAAGTGGTGCTGGGCAAGAAAGGCGAC ACCGTGGAACTGACCTGCACCGCCTCCCAGAAGAAG TCCATCCAGTTCCACTGGAAGAACTCCAACCAGATC AAGATCCTGGGCAACCAGGGCAGCTTCCTGACCAAG GGCCCCTCCAAGCTGAACGACCGGGCCGACTCTCGG AGATCCCTGTGGGATCAGGGCAACTTCCCACTGATC ATCAAGAACCTGAAGATCGAGGACTCCGACACCTAC ATCTGCGAGGTGGAAGATCAGAAAGAAGAGGTGCAG CTGCTGGTGTTCGGCCTGACCGCCAACAGCGACACC CATCTGCTGCAGGGCCAGTCCCTGACCCTGACACTG GAATCTCCACCCGGCTCCAGCCCTTCCGTGCAGTGC AGATCTCCCAGAGGCAAGAACATCCAGGGCGGCAAG ACCCTGTCCGTGTCCCAGCTGGAACTGCAGGACTCT GGCACCTGGACCTGTACCGTGCTGCAGAACCAGAAA AAGGTGGAATTCAAGATCGACATCGTGGTGCTGGCC TTCCAGAAGGCCTCCGGAGGTGGTGGATCCGAGGTG CAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCT GGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGA TTCACCTTCAATAAGTACGCCATGAACTGGGTCCGC CAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGC ATAAGAAGTAAATATAATAATTATGCAACATATTAT GCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGA GATGATTCAAAAAACACTGCCTATCTACAAATGAAC AACTTGAAGACTGAGGACACTGCCGTGTACTACTGT GTGAGACATGGGAACTTCGGTAATAGCTACATATCC TACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACC GTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGC TCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAG GAACCTTCACTCACCGTATCACCTGGTGGAACAGTC ACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACA TCTGGCAACTACCCAAACTGGGTCCAACAAAAACCA GGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAG TTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGC TCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCA GGGGTACAGCCAGAGGATGAGGCAGAATATTACTGT GTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGA GGAACCAAACTGACTGTCCTACATCATCACCATCAT CAT 49 SA25CD4(1 + 2) × artificial AA EDICLPRWGCLWEDKKVVLGKKGDTVELTCTASQKK aCD3 × His tag SIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSR RSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQ LLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQC RSPRGKNIQGGKTLSVSQLELQDSGTWTCTVLQNQK KVEFKIDIVVLAFQKASGGGGSEVQLVESGGGLVQP GGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVAR IRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMN NLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVT VSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTV TLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTK FLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYC VLWYSNRWVFGGGTKLTVLHHHHHH 50 CD4(1 + 2) × aCD3 artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG SA21 × His tag GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCCAGACGGTCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACAGCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACCCTGGAAAGC CCCCCTGGCTCCAGCCCTTCCGTGCAGTGCCGGTCC CCTCGGGGCAAGAACATCCAGGGCGGCAAGACCCTG TCCGTGTCCCAGCTGGAACTGCAGGACAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAATTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAGGCTTCCGGAGGTGGTGGATCCGAGGTGCAGCTG GTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGG TCATTGAAACTCTCATGTGCAGCCTCTGGATTCACC TTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCT CCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGA AGTAAATATAATAATTATGCAACATATTATGCCGAT TCAGTGAAAGACAGGTTCACCATCTCCAGAGATGAT TCAAAAAACACTGCCTATCTACAAATGAACAACTTG AAGACTGAGGACACTGCCGTGTACTACTGTGTGAGA CATGGGAACTTCGGTAATAGCTACATATCCTACTGG GCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCC TCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGT GGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCT TCACTCACCGTATCACCTGGTGGAACAGTCACACTC ACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGC AACTACCCAAACTGGGTCCAACAAAAACCAGGTCAG GCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTC GCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTG CTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTA CAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTA TGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACC AAACTGACTGTCCTACGCCTGATTGAAGATATTTGC CTGCCGCGCTGGGGCTGCCTGTGGGAAGATGATCAT CATCACCATCATCAT 51 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI SA21 × His tag LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLRLIEDICLPRWGCLWEDDHHHHHH 52 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI SA21 LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLRLIEDICLPRWGCLWEDD 53 CD4(1 + 2) × aCD3 artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG LSA21 × His tag GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCCAGACGGTCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACAGCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACCCTGGAAAGC CCCCCTGGCTCCAGCCCTTCCGTGCAGTGCCGGTCC CCTCGGGGCAAGAACATCCAGGGCGGCAAGACCCTG TCCGTGTCCCAGCTGGAACTGCAGGACAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAATTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAGGCTTCCGGAGGCGGAGGATCTGAAGTGCAGCTG GTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA TCTCTGAAGCTGTCTTGTGCCGCCAGCGGCTTCACC TTCAACAAATACGCCATGAACTGGGTGCGACAGGCC CCTGGCAAGGGCCTGGAATGGGTGGCCCGGATCAGA TCCAAGTACAACAACTACGCTACCTACTACGCCGAC TCCGTGAAGGACCGGTTCACCATCTCCCGGGACGAC TCCAAGAACACCGCCTACCTGCAGATGAACAACCTG AAAACCGAGGACACCGCCGTGTACTACTGCGTGCGG CACGGCAACTTCGGCAACTCCTACATCAGCTACTGG GCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCA TCAGGTGGCGGTGGATCTGGGGGAGGCGGTTCAGGC GGAGGGGGATCTCAGACAGTCGTGACCCAGGAACCC TCCCTGACCGTGTCTCCTGGCGGAACCGTGACCCTG ACCTGCGGATCTTCTACCGGCGCTGTGACCTCCGGC AACTACCCTAATTGGGTGCAGCAGAAGCCCGGCCAG GCTCCCAGAGGACTGATCGGCGGCACCAAGTTTCTG GCTCCCGGCACCCCTGCCAGATTCTCCGGTTCTCTG CTGGGCGGCAAGGCCGCTCTGACTCTGTCTGGGGTG CAGCCAGAGGACGAGGCCGAGTACTATTGTGTGCTG TGGTACTCCAACCGCTGGGTGTTCGGCGGAGGCACC AAGCTGACAGTGCTGGGTGGCGGAGGCTCTCGGCTG ATCGAGGACATCTGCCTGCCTAGATGGGGCTGCCTG TGGGAGGACGACCACCACCATCACCACCAC 54 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI LSA21 × His tag LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGSRLIEDICLPRWGCLWEDDHHHHHH 55 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI LSA21 LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGSRLIEDICLPRWGCLWEDD 56 CD4(1 + 2) × aCD3 artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG L × SA21 × His GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC tag CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCCAGACGGTCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACAGCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACCCTGGAAAGC CCCCCTGGCTCCAGCCCTTCCGTGCAGTGCCGGTCC CCTCGGGGCAAGAACATCCAGGGCGGCAAGACCCTG TCCGTGTCCCAGCTGGAACTGCAGGACAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAATTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAGGCTTCCGGAGGCGGAGGATCTGAAGTGCAGCTG GTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA TCTCTGAAGCTGTCTTGTGCCGCCAGCGGCTTCACC TTCAACAAATACGCCATGAACTGGGTGCGACAGGCC CCTGGCAAGGGCCTGGAATGGGTGGCCCGGATCAGA TCCAAGTACAACAACTACGCTACCTACTACGCCGAC TCCGTGAAGGACCGGTTCACCATCTCCCGGGACGAC TCCAAGAACACCGCCTACCTGCAGATGAACAACCTG AAAACCGAGGACACCGCCGTGTACTACTGCGTGCGG CACGGCAACTTCGGCAACTCCTACATCAGCTACTGG GCCTACTGGGGCCAGGGCACCCTCGTGACAGTGTCA TCAGGTGGCGGTGGATCTGGGGGAGGCGGTTCAGGC GGAGGGGGATCTCAGACAGTCGTGACCCAGGAACCC TCCCTGACCGTGTCTCCTGGCGGAACCGTGACCCTG ACCTGCGGATCTTCTACCGGCGCTGTGACCTCCGGC AACTACCCTAATTGGGTGCAGCAGAAGCCCGGCCAG GCTCCCAGAGGACTGATCGGCGGCACCAAGTTTCTG GCTCCCGGCACCCCTGCCAGATTCTCCGGTTCTCTG CTGGGCGGCAAGGCCGCTCTGACTCTGTCTGGGGTG CAGCCAGAGGACGAGGCCGAGTACTATTGTGTGCTG TGGTACTCCAACCGCTGGGTGTTCGGCGGAGGCACC AAGCTGACAGTGCTGGGTGGCGGAGGTTCTGGCGGG GGAGGCAGTGGGGGGGGAGGATCTAGACTGATCGAG GACATCTGCCTGCCCAGATGGGGCTGCCTGTGGGAG GACGATCACCACCACCATCACCAC 57 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI L × SA21 × His LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK tag NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGSGGGGSGGGGSRLIEDICLPRWGCLWE DDHHHHHH 58 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI L × SA21 LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLGGGGSGGGGSGGGGSRLIEDICLPRWGCLWE DD 59 CD4(1 + 2) × aCD3 artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG SA25 × His tag GAACTGACCTGCACCGCCTCCCAGAAGAAGTCCATC CAGTTCCACTGGAAGAACTCCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC TCCAAGCTGAACGACCGGGCCGACTCCAGACGGTCC CTGTGGGATCAGGGCAACTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACTCCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACAGCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACCCTGGAAAGC CCCCCTGGCTCCAGCCCTTCCGTGCAGTGCCGGTCC CCTCGGGGCAAGAACATCCAGGGCGGCAAGACCCTG TCCGTGTCCCAGCTGGAACTGCAGGACAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAATTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAGGCTTCCGGAGGCGGAGGCTCTGAGGTGCAGCTG GTGGAAAGTGGCGGCGGACTGGTGCAGCCTGGCGGC TCCCTGAAGCTGTCTTGCGCCGCCAGCGGCTTCACC TTCAACAAATACGCCATGAACTGGGTCCGACAGGCT CCTGGCAAGGGCCTGGAATGGGTGGCCCGGATTCGG TCCAAGTACAACAACTACGCCACCTACTACGCCGAC TCCGTGAAGGACCGGTTCACCATCAGCCGGGACGAC TCCAAGAACACCGCCTACCTGCAGATGAACAACCTG AAAACCGAGGACACCGCCGTGTACTACTGCGTGCGG CACGGCAACTTCGGCAACTCCTACATCAGCTACTGG GCCTACTGGGGCCAGGGCACCCTGGTGACAGTGTCC TCTGGCGGAGGCGGATCTGGGGGCGGAGGATCAGGC GGGGGAGGATCCCAGACCGTGGTGACACAGGAACCC TCCCTGACCGTCTCTCCTGGGGGCACCGTGACCCTG ACCTGCGGATCTTCCACCGGCGCTGTGACTAGTGGC AACTACCCCAACTGGGTGCAGCAGAAGCCCGGCCAG GCCCCTAGAGGCCTGATCGGCGGCACCAAGTTTCTG GCTCCCGGCACCCCTGCCCGGTTCTCTGGATCTCTG CTGGGCGGCAAGGCCGCTCTGACACTGTCTGGCGTG CAGCCTGAGGACGAGGCCGAGTACTATTGTGTGCTG TGGTACTCCAACAGATGGGTGTTCGGCGGAGGCACC AAGCTGACCGTGCTGGAAGATATCTGCCTGCCCAGA TGGGGCTGCCTGTGGGAGGACCACCACCACCATCAC CAC 60 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI SA25 × His tag LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLEDICLPRWGCLWEDHHHHHH 61 CD4(1 + 2) × aCD3 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI SA25 LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KASGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFT FNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYAD SVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSG GGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSG NYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSL LGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGT KLTVLEDICLPRWGCLWED 62 SA21LCD4(1 + 2) artificial NT CGGCTGATCGAGGACATCTGCCTGCCTAGATGGGGC × aCD3 × His tag TGCCTGTGGGAGGATGATGGCGGCGGAGGCTCCAAG AAAGTGGTGCTGGGCAAGAAAGGCGACACCGTGGAA CTGACCTGCACCGCCTCCCAGAAGAAGTCCATCCAG TTCCACTGGAAGAACTCCAACCAGATCAAGATCCTG GGCAACCAGGGCAGCTTCCTGACCAAGGGCCCCTCC AAGCTGAACGACCGGGCCGACTCTCGGAGATCCCTG TGGGATCAGGGCAACTTCCCACTGATCATCAAGAAC CTGAAGATCGAGGATTCCGACACCTACATCTGCGAG GTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTGGTG TTCGGCCTGACCGCCAACTCCGATACCCATCTGCTG CAGGGCCAGTCCCTGACCCTGACACTGGAATCTCCA CCCGGCTCCAGCCCTTCCGTGCAGTGCAGATCTCCC AGAGGCAAGAACATCCAGGGCGGCAAGACCCTGTCC GTGTCCCAGCTGGAACTGCAGGACTCTGGCACCTGG ACCTGTACCGTGCTGCAGAACCAGAAAAAGGTGGAA TTCAAGATCGACATCGTGGTGCTGGCCTTCCAGAAG GCCTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTC GAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCA TTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTC AATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCA GGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGT AAATATAATAATTATGCAACATATTATGCCGATTCA GTGAAAGACAGGTTCACCATCTCCAGAGATGATTCA AAAAACACTGCCTATCTACAAATGAACAACTTGAAG ACTGAGGACACTGCCGTGTACTACTGTGTGAGACAT GGGAACTTCGGTAATAGCTACATATCCTACTGGGCT TACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGT GGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCA CTCACCGTATCACCTGGTGGAACAGTCACACTCACT TGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAAC TACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCA CCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCC CCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTT GGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAG CCAGAGGATGAGGCAGAATATTACTGTGTTCTATGG TACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAA CTGACTGTCCTACATCATCACCATCATCAT 63 SA21LCD4(1 + 2) artificial AA RLIEDICLPRWGCLWEDDGGGGSKKVVLGKKGDTVE × aCD3 × His tag LTCTASQKKSIQFHWKNSNQIKILGNQGSFLTKGPS KLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICE VEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESP PGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTW TCTVLQNQKKVEFKIDIVVLAFQKASGGGGSEVQLV ESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDS KNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWA YWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPS LTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQA PRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH 64 SA21L × CD4(1 + artificial NT CGGCTGATCGAGGACATCTGCCTGCCTAGATGGGGC 2) × aCD3 × His TGCCTGTGGGAGGATGATGGCGGCGGAGGATCTGGC tag GGAGGTGGAAGCGGAGGGGGCGGATCCAAAAAGGTG GTGCTGGGCAAGAAAGGCGACACCGTGGAACTGACC TGCACCGCCTCCCAGAAGAAGTCCATCCAGTTCCAC TGGAAGAACTCCAACCAGATCAAGATCCTGGGCAAC CAGGGCAGCTTCCTGACCAAGGGCCCCTCCAAGCTG AACGACCGGGCCGACTCTCGGAGATCCCTGTGGGAT CAGGGCAACTTCCCACTGATCATCAAGAACCTGAAG ATCGAGGATTCCGACACCTACATCTGCGAAGTGGAA GATCAGAAAGAAGAGGTGCAGCTGCTGGTGTTCGGC CTGACCGCCAACTCCGATACCCATCTGCTGCAGGGC CAGTCCCTGACCCTGACACTGGAATCTCCACCCGGC TCCAGCCCTTCCGTGCAGTGCAGATCTCCCAGAGGC AAGAACATCCAGGGCGGCAAGACCCTGTCCGTGTCC CAGCTGGAACTGCAGGACTCTGGCACCTGGACCTGT ACCGTGCTGCAGAACCAGAAAAAAGTGGAATTCAAG ATCGACATCGTGGTGCTGGCCTTCCAGAAGGCCTCC GGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCT GGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAA CTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAG TACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAG GGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATAT AATAATTATGCAACATATTATGCCGATTCAGTGAAA GACAGGTTCACCATCTCCAGAGATGATTCAAAAAAC ACTGCCTATCTACAAATGAACAACTTGAAGACTGAG GACACTGCCGTGTACTACTGTGTGAGACATGGGAAC TTCGGTAATAGCTACATATCCTACTGGGCTTACTGG GGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGT GGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGT TCTCAGACTGTTGTGACTCAGGAACCTTCACTCACC GTATCACCTGGTGGAACAGTCACACTCACTTGTGGC TCCTCGACTGGGGCTGTTACATCTGGCAACTACCCA AACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGT GGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGT ACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGC AAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAG GATGAGGCAGAATATTACTGTGTTCTATGGTACAGC AACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACT GTCCTACATCATCACCATCATCAT 65 SA21L × CD4(1 + artificial AA RLIEDICLPRWGCLWEDDGGGGSGGGGSGGGGSKKV 2) × aCD3 × His VLGKKGDTVELTCTASQKKSIQFHWKNSNQIKILGN tag QGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLK IEDSDTYICEVEDQKEEVQLLVFGLTANSDTHLLQG QSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVS QLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKAS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VLHHHHHH 66 CD4(1 + 2)SA21 × artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG aCD3 × His tag GAACTGACCTGCACCGCCAGCCAGAAGAAGTCCATC CAGTTCCACTGGAAGAACAGCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC AGCAAGCTGAACGACAGAGCCGACTCTCGGCGGAGC CTGTGGGACCAGGGCAATTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACAGCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACTCCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACACTGGAAAGC CCTCCAGGCAGCAGCCCCAGCGTGCAGTGTAGAAGC CCCAGAGGCAAGAACATCCAGGGCGGCAAGACCCTG AGCGTGTCCCAGCTGGAACTGCAGGATAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAGTTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAGGCCCGGCTGATCGAGGATATCTGCCTGCCCAGA TGGGGCTGTCTGTGGGAGGACGACGAAGTGCAGCTG GTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA TCTCTGAAGCTGAGCTGTGCCGCCAGCGGCTTCACC TTCAACAAATACGCCATGAACTGGGTGCGCCAGGCC CCTGGCAAAGGCCTGGAATGGGTGGCCCGGATCAGA AGCAAGTACAACAACTATGCCACCTACTACGCCGAC AGCGTGAAGGACCGGTTCACCATCAGCAGGGACGAC TCCAAGAACACCGCCTACCTGCAGATGAACAACCTG AAAACCGAGGATACCGCCGTGTACTACTGCGTGCGG CACGGCAACTTCGGCAACAGCTACATCAGCTACTGG GCCTACTGGGGCCAGGGCACACTCGTGACAGTGTCT AGCGGAGGCGGAGGATCAGGCGGCGGAGGAAGTGGC GGAGGGGGATCTCAGACAGTCGTGACCCAGGAACCC AGCCTGACCGTGTCTCCTGGCGGAACCGTGACACTG ACATGCGGCAGCTCTACAGGCGCCGTGACCAGCGGC AACTACCCTAATTGGGTGCAGCAGAAGCCCGGACAG GCCCCAAGAGGACTGATCGGCGGCACCAAGTTTCTG GCTCCCGGCACCCCTGCCAGATTCAGCGGCTCACTG CTGGGAGGAAAGGCCGCCCTGACTCTGTCTGGGGTG CAGCCAGAGGATGAGGCCGAGTACTATTGTGTGCTG TGGTACAGCAACCGCTGGGTGTTCGGAGGCGGCACA AAGCTGACAGTGCTGCACCACCACCATCACCAC 67 CD4(1 + 2)SA21 × artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI aCD3 × His tag LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KARLIEDICLPRWGCLWEDDEVQLVESGGGLVQPGG SLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIR SKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNL KTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVS SGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTL TCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFL APGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVL WYSNRWVFGGGTKLTVLHHHHHH 68 CD4(1 + 2)LSA21 artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG L × aCD3 × His GAACTGACCTGCACCGCCAGCCAGAAGAAGTCCATC tag CAGTTCCACTGGAAGAACAGCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC AGCAAGCTGAACGACAGAGCCGACTCTCGGCGGAGC CTGTGGGACCAGGGCAATTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACAGCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACTCCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACACTGGAAAGC CCTCCAGGCAGCAGCCCCAGCGTGCAGTGTAGAAGC CCCAGAGGCAAGAACATCCAGGGCGGCAAGACCCTG AGCGTGTCCCAGCTGGAACTGCAGGATAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAGTTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAAGCCGGCGGAGGCGGCTCTAGACTGATCGAGGAT ATCTGCCTGCCCAGATGGGGCTGTCTGTGGGAGGAC GATTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTC GAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCA TTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTC AATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCA GGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGT AAATATAATAATTATGCAACATATTATGCCGATTCA GTGAAAGACAGGTTCACCATCTCCAGAGATGATTCA AAAAACACTGCCTATCTACAAATGAACAACTTGAAG ACTGAGGACACTGCCGTGTACTACTGTGTGAGACAT GGGAACTTCGGTAATAGCTACATATCCTACTGGGCT TACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA GGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGT GGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCA CTCACCGTATCACCTGGTGGAACAGTCACACTCACT TGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAAC TACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCA CCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCC CCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTT GGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAG CCAGAGGATGAGGCAGAATATTACTGTGTTCTATGG TACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAA CTGACTGTCCTACATCATCACCATCATCAT 69 CD4(1 + 2)LSA21 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI L × aCD3 × His LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK tag NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KAGGGGSRLIEDICLPRWGCLWEDDSGGGGSEVQLV ESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAP GKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDS KNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWA YWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPS LTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQA PRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQ PEDEAEYYCVLWYSNRWVFGGGTKLTVLHHHHHH 70 CD4(1 + 2)L × SA2 artificial NT AAGAAAGTGGTGCTGGGCAAGAAAGGCGACACCGTG 1L × aCD3 × His GAACTGACCTGCACCGCCAGCCAGAAGAAGTCCATC tag CAGTTCCACTGGAAGAACAGCAACCAGATCAAGATC CTGGGCAACCAGGGCAGCTTCCTGACCAAGGGCCCC AGCAAGCTGAACGACAGAGCCGACTCTCGGCGGAGC CTGTGGGACCAGGGCAATTTCCCACTGATCATCAAG AACCTGAAGATCGAGGACAGCGACACCTACATCTGC GAGGTGGAAGATCAGAAAGAAGAGGTGCAGCTGCTG GTGTTCGGCCTGACCGCCAACTCCGACACCCATCTG CTGCAGGGCCAGAGCCTGACCCTGACACTGGAAAGC CCTCCAGGCAGCAGCCCCAGCGTGCAGTGTAGAAGC CCCAGAGGCAAGAACATCCAGGGCGGCAAGACCCTG AGCGTGTCCCAGCTGGAACTGCAGGATAGCGGCACC TGGACCTGTACCGTGCTGCAGAACCAGAAAAAGGTG GAGTTCAAGATCGACATCGTGGTGCTGGCCTTCCAG AAAGCCGGCGGAGGCGGATCTGGCGGCGGAGGATCT GGGGGAGGCGGCTCTAGACTGATCGAGGATATCTGC CTGCCCAGATGGGGCTGTCTGTGGGAGGATGATTCT GGCGGAGGGGGAAGTGGGGGGGGAGGATCCGGAGGT GGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGA GGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCA TGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCC ATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTG GAATGGGTTGCTCGCATAAGAAGTAAATATAATAAT TATGCAACATATTATGCCGATTCAGTGAAAGACAGG TTCACCATCTCCAGAGATGATTCAAAAAACACTGCC TATCTACAAATGAACAACTTGAAGACTGAGGACACT GCCGTGTACTACTGTGTGAGACATGGGAACTTCGGT AATAGCTACATATCCTACTGGGCTTACTGGGGCCAA GGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGT TCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAG ACTGTTGTGACTCAGGAACCTTCACTCACCGTATCA CCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCG ACTGGGGCTGTTACATCTGGCAACTACCCAAACTGG GTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTA ATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCT GCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCT GCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAG GCAGAATATTACTGTGTTCTATGGTACAGCAACCGC TGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA CATCATCACCATCATCAT 71 CD4(1 + 2)L × SA2 artificial AA KKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKI 1L × aCD3 × His LGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIK tag NLKIEDSDTYICEVEDQKEEVQLLVFGLTANSDTHL LQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTL SVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQ KAGGGGSGGGGSGGGGSRLIEDICLPRWGCLWEDDS GGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLKLS CAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNN YATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDT AVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGG SGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSS TGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTP ARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNR WVFGGGTKLTVLHHHHHH 72 B12HL artificial NT CAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAG AAACCTGGCGCCTCCGTGAAGGTGTCCTGCCAGGCC TCCGGCTACCGGTTCTCCAACTTCGTGATCCACTGG GTGCGACAGGCCCCTGGCCAGAGATTCGAGTGGATG GGCTGGATCAACCCCTACAACGGCAACAAAGAGTTC TCCGCCAAGTTCCAGGACAGAGTGACCTTCACCGCC GACACCTCCGCCAACACCGCCTACATGGAACTGCGG TCCCTGAGAAGCGCCGACACCGCCGTGTACTACTGC GCCAGAGTGGGCCCCTACTCCTGGGACGACTCCCCC CAGGACAACTACTACATGGACGTGTGGGGCAAGGGC ACCACCGTGATCGTGTCCTCTGGCGGCGGAGGATCT GGCGGAGGCGGAAGTGGCGGAGGGGGCTCTGAGATC GTGCTGACCCAGTCCCCCGGCACACTGTCTCTGAGC CCTGGCGAGCGGGCCACCTTCTCTTGCCGGTCCTCC CACTCCATCCGGTCCAGACGGGTGGCCTGGTATCAG CACAAGCCAGGCCAGGCTCCTCGGCTGGTGATCCAC GGCGTGTCCAACCGGGCCTCCGGCATCTCCGACAGA TTCAGCGGCTCCGGCAGCGGCACCGACTTCACCCTG ACCATCACCCGCGTGGAACCCGAGGACTTCGCCCTG TACTATTGCCAGGTGTACGGCGCCTCCTCCTACACC TTCGGCCAGGGCACTAAGCTGGAACGGAAG 73 B12HL artificial AA QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHW VRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTA DTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSP QDNYYMDVWGKGTTVIVSSGGGGSGGGGSGGGGSEI VLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQ HKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTL TITRVEPEDFALYYCQVYGASSYTFGQGTKLERK 74 B12HL × aCD3 × artificial NT CAAGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAG SA21 × His tag AAACCTGGCGCCTCCGTGAAGGTGTCCTGCCAGGCC TCCGGCTACCGGTTCTCCAACTTCGTGATCCACTGG GTGCGACAGGCCCCTGGCCAGAGATTCGAGTGGATG GGCTGGATCAACCCCTACAACGGCAACAAAGAGTTC TCCGCCAAGTTCCAGGACAGAGTGACCTTCACCGCC GACACCTCCGCCAACACCGCCTACATGGAACTGCGG TCCCTGAGAAGCGCCGACACCGCCGTGTACTACTGC GCCAGAGTGGGCCCCTACTCCTGGGACGACTCCCCC CAGGACAACTACTACATGGACGTGTGGGGCAAGGGC ACCACCGTGATCGTGTCCTCTGGCGGCGGAGGATCT GGCGGAGGCGGAAGTGGCGGAGGGGGCTCTGAGATC GTGCTGACCCAGTCCCCCGGCACACTGTCTCTGAGC CCTGGCGAGCGGGCCACCTTCTCTTGCCGGTCCTCC CACTCCATCCGGTCCAGACGGGTGGCCTGGTATCAG CACAAGCCAGGCCAGGCTCCTCGGCTGGTGATCCAC GGCGTGTCCAACCGGGCCTCCGGCATCTCCGACAGA TTCAGCGGCTCCGGCAGCGGCACCGACTTCACCCTG ACCATCACCCGCGTGGAACCCGAGGACTTCGCCCTG TACTATTGCCAGGTGTACGGCGCCTCCTCCTACACC TTCGGCCAGGGCACTAAGCTGGAACGGAAGTCCGGA GGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGA GGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTC TCATGTGCAGCCTCTGGATTCACCTTCAATAAGTAC GCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGT TTGGAATGGGTTGCTCGCATAAGAAGTAAATATAAT AATTATGCAACATATTATGCCGATTCAGTGAAAGAC AGGTTCACCATCTCCAGAGATGATTCAAAAAACACT GCCTATCTACAAATGAACAACTTGAAGACTGAGGAC ACTGCCGTGTACTACTGTGTGAGACATGGGAACTTC GGTAATAGCTACATATCCTACTGGGCTTACTGGGGC CAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGT GGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCT CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTA TCACCTGGTGGAACAGTCACACTCACTTGTGGCTCC TCGACTGGGGCTGTTACATCTGGCAACTACCCAAAC TGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGT CTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACT CCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAG GCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGAT GAGGCAGAATATTACTGTGTTCTATGGTACAGCAAC CGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTC CTACGCCTGATTGAAGATATTTGCCTGCCGCGCTGG GGCTGCCTGTGGGAAGATGATCATCATCACCATCAT CAT 75 B12HL × aCD3 × artificial AA QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHW SA21 × His tag VRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTA DTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSP QDNYYMDVWGKGTTVIVSSGGGGSGGGGSGGGGSEI VLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQ HKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTL TITRVEPEDFALYYCQVYGASSYTFGQGTKLERKSG GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNF GNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGS QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV LRLIEDICLPRWGCLWEDDHHHHHH 76 B12HL × aCD3 × artificial AA QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHW SA21 VRQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTA DTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSP QDNYYMDVWGKGTTVIVSSGGGGSGGGGSGGGGSEI VLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQ HKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTL TITRVEPEDFALYYCQVYGASSYTFGQGTKLERKSG GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNF GNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGS QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV LRLIEDICLPRWGCLWEDD 77 B12LH artificial NT GAGATCGTGCTGACCCAGTCCCCCGGCACACTGTCT CTGAGCCCTGGCGAGCGGGCCACCTTCTCTTGCCGG TCCTCCCACTCCATCCGGTCCAGACGGGTGGCCTGG TATCAGCACAAGCCAGGCCAGGCCCCTCGGCTGGTG ATCCACGGCGTGTCCAACCGGGCCTCCGGCATCTCC GACAGATTCTCCGGCTCCGGCAGCGGCACCGACTTC ACCCTGACCATCACCCGCGTGGAACCCGAGGACTTC GCCCTGTACTACTGCCAGGTGTACGGCGCCTCCTCC TACACCTTCGGCCAGGGCACCAAGCTGGAAAGAAAG GGCGGAGGCGGCTCTGGTGGCGGAGGAAGTGGAGGC GGAGGATCTCAGGTGCAGCTGGTGCAGTCTGGCGCC GAAGTGAAGAAACCTGGCGCCTCCGTGAAGGTGTCC TGCCAGGCCAGCGGCTACCGGTTCTCCAACTTCGTG ATCCACTGGGTGCGACAGGCACCTGGCCAGAGATTC GAGTGGATGGGCTGGATCAACCCCTACAACGGCAAC AAAGAGTTCTCCGCCAAGTTCCAGGACAGAGTGACC TTCACCGCCGACACCTCCGCCAACACCGCCTACATG GAACTGCGGTCCCTGAGAAGCGCCGACACCGCTGTG TACTACTGTGCCAGAGTGGGCCCCTACTCCTGGGAC GACTCCCCCCAGGACAACTACTACATGGACGTGTGG GGCAAGGGCACTACCGTGATCGTGTCTTCC 78 B12LH artificial AA EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAW YQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDF TLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS CQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGN KEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAV YYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSS 79 B12LH × aCD3 × artificial NT GAGATCGTGCTGACCCAGTCCCCCGGCACACTGTCT SA21 × His tag CTGAGCCCTGGCGAGCGGGCCACCTTCTCTTGCCGG TCCTCCCACTCCATCCGGTCCAGACGGGTGGCCTGG TATCAGCACAAGCCAGGCCAGGCCCCTCGGCTGGTG ATCCACGGCGTGTCCAACCGGGCCTCCGGCATCTCC GACAGATTCTCCGGCTCCGGCAGCGGCACCGACTTC ACCCTGACCATCACCCGCGTGGAACCCGAGGACTTC GCCCTGTACTACTGCCAGGTGTACGGCGCCTCCTCC TACACCTTCGGCCAGGGCACCAAGCTGGAAAGAAAG GGCGGAGGCGGCTCTGGTGGCGGAGGAAGTGGAGGC GGAGGATCTCAGGTGCAGCTGGTGCAGTCTGGCGCC GAAGTGAAGAAACCTGGCGCCTCCGTGAAGGTGTCC TGCCAGGCCAGCGGCTACCGGTTCTCCAACTTCGTG ATCCACTGGGTGCGACAGGCACCTGGCCAGAGATTC GAGTGGATGGGCTGGATCAACCCCTACAACGGCAAC AAAGAGTTCTCCGCCAAGTTCCAGGACAGAGTGACC TTCACCGCCGACACCTCCGCCAACACCGCCTACATG GAACTGCGGTCCCTGAGAAGCGCCGACACCGCTGTG TACTACTGTGCCAGAGTGGGCCCCTACTCCTGGGAC GACTCCCCCCAGGACAACTACTACATGGACGTGTGG GGCAAGGGCACTACCGTGATCGTGTCTTCCGGAGGT GGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGA GGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCA TGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCC ATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTG GAATGGGTTGCTCGCATAAGAAGTAAATATAATAAT TATGCAACATATTATGCCGATTCAGTGAAAGACAGG TTCACCATCTCCAGAGATGATTCAAAAAACACTGCC TATCTACAAATGAACAACTTGAAGACTGAGGACACT GCCGTGTACTACTGTGTGAGACATGGGAACTTCGGT AATAGCTACATATCCTACTGGGCTTACTGGGGCCAA GGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGT TCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAG ACTGTTGTGACTCAGGAACCTTCACTCACCGTATCA CCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCG ACTGGGGCTGTTACATCTGGCAACTACCCAAACTGG GTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTA ATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCT GCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCT GCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAG GCAGAATATTACTGTGTTCTATGGTACAGCAACCGC TGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA CGCCTGATTGAAGATATTTGCCTGCCGCGCTGGGGC TGCCTGTGGGAAGATGATCATCATCACCATCATCAT 80 B12LH × aCD3 × S artificial AA EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAW A21 × His tag YQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDF TLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS CQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGN KEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAV YYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL RLIEDICLPRWGCLWEDDHHHHHH 81 B12LH × aCD3 × S artificial AA EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAW A21 YQHKPGQAPRLVIHGVSNRASGISDRFSGSGSGTDF TLTITRVEPEDFALYYCQVYGASSYTFGQGTKLERK GGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS CQASGYRFSNFVIHWVRQAPGQRFEWMGWINPYNGN KEFSAKFQDRVTFTADTSANTAYMELRSLRSADTAV YYCARVGPYSWDDSPQDNYYMDVWGKGTTVIVSSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL RLIEDICLPRWGCLWEDD 82 VRC01HL artificial NT CAGGTGCAGCTGGTGCAGTCTGGCGGCCAGATGAAG AAACCCGGCGAGAGCATGCGGATCAGCTGCCGGGCC TCTGGATACGAGTTCATCGACAGCACTCTGAACTGG ATCCGGCTGGCCCCTGGCAAGAGGCCCGAGTGGATG GGCTGGCTGAAGCCCAGAGGCGGAGCCGTGAACTAC GCCAGACCTCTGCAGGGCAGAGTGACCATGACCCGG GACGTCTACAGCGATACCGCCTTCCTGGAACTGCGG AGCCTGACCGTGGACGATACCGCCGTGTACTTCTGT ACTCGGGGCAAGAACGCCGACTACAACTGGGACTTC GAGCACTGGGGCAGAGGCACCCCCGTGATCGTGTCT AGCGGAGGCGGAGGATCTGGCGGCGGAGGCTCTGGG GGAGGCGGAAGCGAGATCGTGCTGACCCAGAGCCCT GGCACCCTGAGCCTGTCTCCCGGCGAAACCGCCATC ATCAGCTGCAGAACCAGCCAGTACGGCAGCCTGGCC TGGTATCAGCAGAGGCCAGGCCAGGCCCCCAGACTC GTGATCTACAGTGGAAGCACCAGAGCCGCCGGAATC CCCGACCGGTTCTCTGGTTCCAGATGGGGCCCTGAC TACAACCTGACCATCAGCAACCTGGAAAGCGGCGAC TTCGGCGTGTACTACTGCCAGCAGTACGAGTTCTTC GGCCAGGGCACCAAGGTCCAGGTGGACATCAAG 83 VRC01HL artificial AA QVQLVQSGGQMKKPGESMRISCRASGYEFIDSTLNW IRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTR DVYSDTAFLELRSLTVDDTAVYFCTRGKNADYNWDF EHWGRGTPVIVSSGGGGSGGGGSGGGGSEIVLTQSP GTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRL VIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGD FGVYYCQQYEFFGQGTKVQVDIK 84 VRC01HL × aCD artificial NT CAGGTGCAGCTGGTGCAGTCTGGCGGCCAGATGAAG 3 × SA21 × His AAACCCGGCGAGAGCATGCGGATCAGCTGCCGGGCC tag TCTGGATACGAGTTCATCGACAGCACTCTGAACTGG ATCCGGCTGGCCCCTGGCAAGAGGCCCGAGTGGATG GGCTGGCTGAAGCCCAGAGGCGGAGCCGTGAACTAC GCCAGACCTCTGCAGGGCAGAGTGACCATGACCCGG GACGTCTACAGCGATACCGCCTTCCTGGAACTGCGG AGCCTGACCGTGGACGATACCGCCGTGTACTTCTGT ACTCGGGGCAAGAACGCCGACTACAACTGGGACTTC GAGCACTGGGGCAGAGGCACCCCCGTGATCGTGTCT AGCGGAGGCGGAGGATCTGGCGGCGGAGGCTCTGGG GGAGGCGGAAGCGAGATCGTGCTGACCCAGAGCCCT GGCACCCTGAGCCTGTCTCCCGGCGAAACCGCCATC ATCAGCTGCAGAACCAGCCAGTACGGCAGCCTGGCC TGGTATCAGCAGAGGCCAGGCCAGGCCCCCAGACTC GTGATCTACAGTGGAAGCACCAGAGCCGCCGGAATC CCCGACCGGTTCTCTGGTTCCAGATGGGGCCCTGAC TACAACCTGACCATCAGCAACCTGGAAAGCGGCGAC TTCGGCGTGTACTACTGCCAGCAGTACGAGTTCTTC GGCCAGGGCACCAAGGTCCAGGTGGACATCAAGTCC GGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCT GGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAA CTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAG TACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAG GGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATAT AATAATTATGCAACATATTATGCCGATTCAGTGAAA GACAGGTTCACCATCTCCAGAGATGATTCAAAAAAC ACTGCCTATCTACAAATGAACAACTTGAAGACTGAG GACACTGCCGTGTACTACTGTGTGAGACATGGGAAC TTCGGTAATAGCTACATATCCTACTGGGCTTACTGG GGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGT GGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGT TCTCAGACTGTTGTGACTCAGGAACCTTCACTCACC GTATCACCTGGTGGAACAGTCACACTCACTTGTGGC TCCTCGACTGGGGCTGTTACATCTGGCAACTACCCA AACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGT GGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGT ACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGC AAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAG GATGAGGCAGAATATTACTGTGTTCTATGGTACAGC AACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACT GTCCTACGCCTGATTGAAGATATTTGCCTGCCGCGC TGGGGCTGCCTGTGGGAAGATGATCATCATCACCAT CATCAT 85 VRC01HL × aCD artificial AA QVQLVQSGGQMKKPGESMRISCRASGYEFIDSTLNW 3 × SA21 × His IRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTR tag DVYSDTAFLELRSLTVDDTAVYFCTRGKNADYNWDF EHWGRGTPVIVSSGGGGSGGGGSGGGGSEIVLTQSP GTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRL VIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGD FGVYYCQQYEFFGQGTKVQVDIKSGGGGSEVQLVES GGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKN TAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYW GQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLT VSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVLRLIEDICLPR WGCLWEDDHHHHHH 86 VRC01HL × aCD artificial AA QVQLVQSGGQMKKPGESMRISCRASGYEFIDSTLNW 3 × SA21 IRLAPGKRPEWMGWLKPRGGAVNYARPLQGRVTMTR DVYSDTAFLELRSLTVDDTAVYFCTRGKNADYNWDF EHWGRGTPVIVSSGGGGSGGGGSGGGGSEIVLTQSP GTLSLSPGETAIISCRTSQYGSLAWYQQRPGQAPRL VIYSGSTRAAGIPDRFSGSRWGPDYNLTISNLESGD FGVYYCQQYEFFGQGTKVQVDIKSGGGGSEVQLVES GGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGK GLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKN TAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYW GQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLT VSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPR GLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPE DEAEYYCVLWYSNRWVFGGGTKLTVLRLIEDICLPR WGCLWEDD 87 VRC01LH artificial NT GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGC CTGTCTCCAGGCGAGACAGCCATCATCAGCTGCCGG ACCAGCCAGTACGGCAGCCTGGCTTGGTATCAGCAG AGGCCAGGACAGGCCCCCAGACTCGTGATCTACTCT GGAAGCACCAGAGCCGCCGGAATCCCCGACCGGTTC TCTGGATCCCGCTGGGGCCCTGACTACAACCTGACC ATCAGCAACCTGGAAAGCGGCGACTTCGGCGTGTAC TACTGCCAGCAGTACGAGTTCTTCGGCCAGGGCACC AAGGTCCAGGTGGACATCAAGGGCGGAGGCGGATCT GGCGGAGGAGGAAGCGGAGGCGGAGGATCTCAGGTG CAGCTGGTGCAGTCTGGCGGCCAGATGAAGAAACCC GGCGAGAGCATGCGGATCAGCTGCAGAGCCTCTGGA TACGAGTTCATCGACAGCACTCTGAACTGGATCCGG CTGGCCCCTGGCAAGAGGCCCGAGTGGATGGGCTGG CTGAAGCCCAGAGGCGGAGCCGTGAACTACGCCAGA CCTCTGCAGGGCAGAGTGACCATGACCCGGGACGTC TACAGCGATACCGCCTTCCTGGAACTGCGGAGCCTG ACCGTGGACGATACCGCCGTGTACTTCTGTACTCGG GGCAAGAACGCCGACTACAACTGGGACTTCGAGCAC TGGGGCAGAGGCACCCCCGTGATCGTGTCCTCC 88 VRC01LH artificial AA EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQ RPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLT ISNLESGDFGVYYCQQYEFFGQGTKVQVDIKGGGGS GGGGSGGGGSQVQLVQSGGQMKKPGESMRISCRASG YEFIDSTLNWIRLAPGKRPEWMGWLKPRGGAVNYAR PLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTR GKNADYNWDFEHWGRGTPVIVSS 89 VRC01LH × aCD artificial NT GAGATCGTGCTGACCCAGAGCCCCGGCACCCTGAGC 3 × SA21 × His CTGTCTCCAGGCGAGACAGCCATCATCAGCTGCCGG tag ACCAGCCAGTACGGCAGCCTGGCTTGGTATCAGCAG AGGCCAGGACAGGCCCCCAGACTCGTGATCTACTCT GGAAGCACCAGAGCCGCCGGAATCCCCGACCGGTTC TCTGGATCCCGCTGGGGCCCTGACTACAACCTGACC ATCAGCAACCTGGAAAGCGGCGACTTCGGCGTGTAC TACTGCCAGCAGTACGAGTTCTTCGGCCAGGGCACC AAGGTCCAGGTGGACATCAAGGGCGGAGGCGGATCT GGCGGAGGAGGAAGCGGAGGCGGAGGATCTCAGGTG CAGCTGGTGCAGTCTGGCGGCCAGATGAAGAAACCC GGCGAGAGCATGCGGATCAGCTGCAGAGCCTCTGGA TACGAGTTCATCGACAGCACTCTGAACTGGATCCGG CTGGCCCCTGGCAAGAGGCCCGAGTGGATGGGCTGG CTGAAGCCCAGAGGCGGAGCCGTGAACTACGCCAGA CCTCTGCAGGGCAGAGTGACCATGACCCGGGACGTC TACAGCGATACCGCCTTCCTGGAACTGCGGAGCCTG ACCGTGGACGATACCGCCGTGTACTTCTGTACTCGG GGCAAGAACGCCGACTACAACTGGGACTTCGAGCAC TGGGGCAGAGGCACCCCCGTGATCGTGTCCTCCGGA GGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGA GGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTC TCATGTGCAGCCTCTGGATTCACCTTCAATAAGTAC GCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGT TTGGAATGGGTTGCTCGCATAAGAAGTAAATATAAT AATTATGCAACATATTATGCCGATTCAGTGAAAGAC AGGTTCACCATCTCCAGAGATGATTCAAAAAACACT GCCTATCTACAAATGAACAACTTGAAGACTGAGGAC ACTGCCGTGTACTACTGTGTGAGACATGGGAACTTC GGTAATAGCTACATATCCTACTGGGCTTACTGGGGC CAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGT GGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCT CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTA TCACCTGGTGGAACAGTCACACTCACTTGTGGCTCC TCGACTGGGGCTGTTACATCTGGCAACTACCCAAAC TGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGT CTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACT CCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAG GCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGAT GAGGCAGAATATTACTGTGTTCTATGGTACAGCAAC CGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTC CTACGCCTGATTGAAGATATTTGCCTGCCGCGCTGG GGCTGCCTGTGGGAAGATGATCATCATCACCATCAT CAT 90 VRC01LH × aCD artificial AA EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQ 3 × SA21 × His RPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLT tag ISNLESGDFGVYYCQQYEFFGQGTKVQVDIKGGGGS GGGGSGGGGSQVQLVQSGGQMKKPGESMRISCRASG YEFIDSTLNWIRLAPGKRPEWMGWLKPRGGAVNYAR PLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTR GKNADYNWDFEHWGRGTPVIVSSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTV SPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYSNRWVFGGGTKLTVLRLIEDICLPRW GCLWEDDHHHHHH 91 VRC01LH × aCD artificial AA EIVLTQSPGTLSLSPGETAIISCRTSQYGSLAWYQQ 3 × SA21 RPGQAPRLVIYSGSTRAAGIPDRFSGSRWGPDYNLT ISNLESGDFGVYYCQQYEFFGQGTKVQVDIKGGGGS GGGGSGGGGSQVQLVQSGGQMKKPGESMRISCRASG YEFIDSTLNWIRLAPGKRPEWMGWLKPRGGAVNYAR PLQGRVTMTRDVYSDTAFLELRSLTVDDTAVYFCTR GKNADYNWDFEHWGRGTPVIVSSGGGGSEVQLVESG GGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNT AYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWG QGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTV SPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRG LIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYSNRWVFGGGTKLTVLRLIEDICLPRW GCLWEDD 92 4E10HL artificial NT CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAA AGACCCGGCAGCAGCGTGACCGTGTCCTGCAAAGCT AGCGGCGGCAGCTTCAGCACCTACGCCCTGTCTTGG GTGCGCCAGGCTCCTGGCAGAGGCCTGGAATGGATG GGCGGAGTGATCCCCCTGCTGACCATCACCAACTAC GCCCCCAGATTCCAGGGCCGGATCACCATCACCGCC GACAGAAGCACCAGCACCGCCTACCTGGAACTGAAC AGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGT GCCAGAGAGGGCACAACAGGCTGGGGCTGGCTGGGC AAACCTATCGGAGCCTTTGCCCACTGGGGCCAGGGC ACACTCGTGACAGTGTCTAGTGGCGGCGGAGGATCT GGCGGAGGCGGAAGTGGGGGAGGCGGCTCTGAAATC GTGCTGACACAGAGCCCAGGCACCCAGTCTCTGAGC CCTGGCGAAAGAGCCACCCTGAGCTGTAGAGCCAGC CAGAGCGTGGGCAACAACAAGCTGGCCTGGTATCAG CAGCGGCCAGGCCAGGCACCTCGGCTGCTGATCTAT GGCGCCAGCAGCAGACCTAGCGGCGTGGCCGATAGA TTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTG ACCATCTCCAGACTGGAACCCGAGGACTTTGCCGTG TATTATTGCCAGCAGTACGGCCAGAGCCTGAGCACC TTTGGACAGGGCACCAAGGTGGAAGTGAAG 93 4E10HL artificial AA QVQLVQSGAEVKRPGSSVTVSCKASGGSFSTYALSW VRQAPGRGLEWMGGVIPLLTITNYAPRFQGRITITA DRSTSTAYLELNSLRPEDTAVYYCAREGTTGWGWLG KPIGAFAHWGQGTLVTVSSGGGGSGGGGSGGGGSEI VLTQSPGTQSLSPGERATLSCRASQSVGNNKLAWYQ QRPGQAPRLLIYGASSRPSGVADRFSGSGSGTDFTL TISRLEPEDFAVYYCQQYGQSLSTFGQGTKVEVK 94 4E10HL × aCD3 × artificial NT CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAA SA21 × His tag AGACCCGGCAGCAGCGTGACCGTGTCCTGCAAAGCT AGCGGCGGCAGCTTCAGCACCTACGCCCTGTCTTGG GTGCGCCAGGCTCCTGGCAGAGGCCTGGAATGGATG GGCGGAGTGATCCCCCTGCTGACCATCACCAACTAC GCCCCCAGATTCCAGGGCCGGATCACCATCACCGCC GACAGAAGCACCAGCACCGCCTACCTGGAACTGAAC AGCCTGAGGCCCGAGGACACCGCCGTGTACTACTGT GCCAGAGAGGGCACAACAGGCTGGGGCTGGCTGGGC AAACCTATCGGAGCCTTTGCCCACTGGGGCCAGGGC ACACTCGTGACAGTGTCTAGTGGCGGCGGAGGATCT GGCGGAGGCGGAAGTGGGGGAGGCGGCTCTGAAATC GTGCTGACACAGAGCCCAGGCACCCAGTCTCTGAGC CCTGGCGAAAGAGCCACCCTGAGCTGTAGAGCCAGC CAGAGCGTGGGCAACAACAAGCTGGCCTGGTATCAG CAGCGGCCAGGCCAGGCACCTCGGCTGCTGATCTAT GGCGCCAGCAGCAGACCTAGCGGCGTGGCCGATAGA TTTTCCGGCTCTGGCAGCGGCACCGACTTCACCCTG ACCATCTCCAGACTGGAACCCGAGGACTTTGCCGTG TATTATTGCCAGCAGTACGGCCAGAGCCTGAGCACC TTTGGACAGGGCACCAAGGTGGAAGTGAAGTCCGGA GGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGA GGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTC TCATGTGCAGCCTCTGGATTCACCTTCAATAAGTAC GCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGT TTGGAATGGGTTGCTCGCATAAGAAGTAAATATAAT AATTATGCAACATATTATGCCGATTCAGTGAAAGAC AGGTTCACCATCTCCAGAGATGATTCAAAAAACACT GCCTATCTACAAATGAACAACTTGAAGACTGAGGAC ACTGCCGTGTACTACTGTGTGAGACATGGGAACTTC GGTAATAGCTACATATCCTACTGGGCTTACTGGGGC CAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGT GGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCT CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTA TCACCTGGTGGAACAGTCACACTCACTTGTGGCTCC TCGACTGGGGCTGTTACATCTGGCAACTACCCAAAC TGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGT CTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACT CCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAG GCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGAT GAGGCAGAATATTACTGTGTTCTATGGTACAGCAAC CGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTC CTACGCCTGATTGAAGATATTTGCCTGCCGCGCTGG GGCTGCCTGTGGGAAGATGATCATCATCACCATCAT CAT 95 4E10HL × aCD3 × artificial AA QVQLVQSGAEVKRPGSSVTVSCKASGGSFSTYALSW SA21 × His tag VRQAPGRGLEWMGGVIPLLTITNYAPRFQGRITITA DRSTSTAYLELNSLRPEDTAVYYCAREGTTGWGWLG KPIGAFAHWGQGTLVTVSSGGGGSGGGGSGGGGSEI VLTQSPGTQSLSPGERATLSCRASQSVGNNKLAWYQ QRPGQAPRLLIYGASSRPSGVADRFSGSGSGTDFTL TISRLEPEDFAVYYCQQYGQSLSTFGQGTKVEVKSG GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNF GNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGS QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV LRLIEDICLPRWGCLWEDDHHHHHH 96 4E10HL × aCD3 × artificial AA QVQLVQSGAEVKRPGSSVTVSCKASGGSFSTYALSW SA21 VRQAPGRGLEWMGGVIPLLTITNYAPRFQGRITITA DRSTSTAYLELNSLRPEDTAVYYCAREGTTGWGWLG KPIGAFAHWGQGTLVTVSSGGGGSGGGGSGGGGSEI VLTQSPGTQSLSPGERATLSCRASQSVGNNKLAWYQ QRPGQAPRLLIYGASSRPSGVADRFSGSGSGTDFTL TISRLEPEDFAVYYCQQYGQSLSTFGQGTKVEVKSG GGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKY AMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNF GNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGS QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV LRLIEDICLPRWGCLWEDD 97 4E10LH artificial NT GAAATCGTGCTGACACAGAGCCCAGGCACCCAGTCT CTGAGCCCTGGCGAAAGAGCCACCCTGAGCTGTAGA GCCAGCCAGAGCGTGGGCAACAACAAGCTGGCCTGG TATCAGCAGCGGCCAGGCCAGGCACCTCGGCTGCTG ATCTATGGCGCCAGCAGCAGACCTAGCGGCGTGGCC GATAGATTTTCCGGCTCTGGCAGCGGCACCGACTTC ACCCTGACCATCTCCAGACTGGAACCCGAGGACTTT GCCGTGTATTATTGCCAGCAGTACGGCCAGAGCCTG AGCACCTTTGGACAGGGCACCAAGGTGGAAGTGAAG GGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGGGGA GGCGGCTCTCAGGTGCAGCTGGTGCAGTCTGGCGCC GAAGTGAAAAGACCCGGCAGCAGCGTGACCGTGTCC TGCAAAGCTAGCGGCGGCAGCTTCAGCACCTACGCC CTGTCTTGGGTGCGCCAGGCTCCTGGCAGAGGCCTG GAATGGATGGGCGGAGTGATCCCCCTGCTGACCATC ACCAACTACGCCCCCAGATTCCAGGGCCGGATCACC ATCACCGCCGACAGAAGCACCAGCACCGCCTACCTG GAACTGAACAGCCTGAGGCCCGAGGACACCGCCGTG TACTACTGTGCCAGAGAGGGCACAACAGGCTGGGGC TGGCTGGGCAAACCTATCGGAGCCTTTGCCCACTGG GGCCAGGGCACACTCGTGACAGTGTCTTCC 98 4E10LH artificial AA EIVLTQSPGTQSLSPGERATLSCRASQSVGNNKLAW YQQRPGQAPRLLIYGASSRPSGVADRFSGSGSGTDF TLTISRLEPEDFAVYYCQQYGQSLSTFGQGTKVEVK GGGGSGGGGSGGGGSQVQLVQSGAEVKRPGSSVTVS CKASGGSFSTYALSWVRQAPGRGLEWMGGVIPLLTI TNYAPRFQGRITITADRSTSTAYLELNSLRPEDTAV YYCAREGTTGWGWLGKPIGAFAHWGQGTLVTVSS 99 4E10LH × aCD3S artificial NT GAAATCGTGCTGACACAGAGCCCAGGCACCCAGTCT A21 × His tag CTGAGCCCTGGCGAAAGAGCCACCCTGAGCTGTAGA GCCAGCCAGAGCGTGGGCAACAACAAGCTGGCCTGG TATCAGCAGCGGCCAGGCCAGGCACCTCGGCTGCTG ATCTATGGCGCCAGCAGCAGACCTAGCGGCGTGGCC GATAGATTTTCCGGCTCTGGCAGCGGCACCGACTTC ACCCTGACCATCTCCAGACTGGAACCCGAGGACTTT GCCGTGTATTATTGCCAGCAGTACGGCCAGAGCCTG AGCACCTTTGGACAGGGCACCAAGGTGGAAGTGAAG GGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGGGGA GGCGGCTCTCAGGTGCAGCTGGTGCAGTCTGGCGCC GAAGTGAAAAGACCCGGCAGCAGCGTGACCGTGTCC TGCAAAGCTAGCGGCGGCAGCTTCAGCACCTACGCC CTGTCTTGGGTGCGCCAGGCTCCTGGCAGAGGCCTG GAATGGATGGGCGGAGTGATCCCCCTGCTGACCATC ACCAACTACGCCCCCAGATTCCAGGGCCGGATCACC ATCACCGCCGACAGAAGCACCAGCACCGCCTACCTG GAACTGAACAGCCTGAGGCCCGAGGACACCGCCGTG TACTACTGTGCCAGAGAGGGCACAACAGGCTGGGGC TGGCTGGGCAAACCTATCGGAGCCTTTGCCCACTGG GGCCAGGGCACACTCGTGACAGTGTCTTCCGGAGGT GGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGA GGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCA TGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCC ATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTG GAATGGGTTGCTCGCATAAGAAGTAAATATAATAAT TATGCAACATATTATGCCGATTCAGTGAAAGACAGG TTCACCATCTCCAGAGATGATTCAAAAAACACTGCC TATCTACAAATGAACAACTTGAAGACTGAGGACACT GCCGTGTACTACTGTGTGAGACATGGGAACTTCGGT AATAGCTACATATCCTACTGGGCTTACTGGGGCCAA GGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGT TCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAG ACTGTTGTGACTCAGGAACCTTCACTCACCGTATCA CCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCG ACTGGGGCTGTTACATCTGGCAACTACCCAAACTGG GTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTA ATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCT GCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCT GCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAG GCAGAATATTACTGTGTTCTATGGTACAGCAACCGC TGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA CGCCTGATTGAAGATATTTGCCTGCCGCGCTGGGGC TGCCTGTGGGAAGATGATCATCATCACCATCATCAT 100 4E10LH × aCD3S artificial AA EIVLTQSPGTQSLSPGERATLSCRASQSVGNNKLAW A21 × His tag YQQRPGQAPRLLIYGASSRPSGVADRFSGSGSGTDF TLTISRLEPEDFAVYYCQQYGQSLSTFGQGTKVEVK GGGGSGGGGSGGGGSQVQLVQSGAEVKRPGSSVTVS CKASGGSFSTYALSWVRQAPGRGLEWMGGVIPLLTI TNYAPRFQGRITITADRSTSTAYLELNSLRPEDTAV YYCAREGTTGWGWLGKPIGAFAHWGQGTLVTVSSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL RLIEDICLPRWGCLWEDDHHHHHH 101 4E10LH × aCD3S artificial AA EIVLTQSPGTQSLSPGERATLSCRASQSVGNNKLAW A21 YQQRPGQAPRLLIYGASSRPSGVADRFSGSGSGTDF TLTISRLEPEDFAVYYCQQYGQSLSTFGQGTKVEVK GGGGSGGGGSGGGGSQVQLVQSGAEVKRPGSSVTVS CKASGGSFSTYALSWVRQAPGRGLEWMGGVIPLLTI TNYAPRFQGRITITADRSTSTAYLELNSLRPEDTAV YYCAREGTTGWGWLGKPIGAFAHWGQGTLVTVSSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL RLIEDICLPRWGCLWEDD