CL and/or CH1 mutated antibodies for drug conjugation

Abstract

Antibodies having modified constant regions so as to permit conjugation of the antibody to a payload such as a therapeutic agent are described. Preferred antibodies include a mutation at light chain position 180 (positional numbering), most preferably the mutation is to a residue selected from C, K, Q, or a non-natural amino acid. Additional mutations may also be combined with a mutation at position 180; including one or more of light chain (LC) S208, LC S171, LC S182, LC A184, LC V191, LC S202, LC S203, LC T206, heavy chain (HC) S160, HC T190, HC S443, HC S447, HC S139, HC S168, HC V170, HC V176, HC T200, HC S445 according to a positional numbering convention.

Claims

1. An antibody, or a fragment or derivative thereof, having a variable region which binds a target molecule, and a constant region, wherein the constant region comprises one or more mutations introducing a site specific conjugation site selected so as to permit conjugation of the antibody, fragment, or derivative to a payload, wherein the antibody comprises the amino acid sequence of SEQ ID NO: 36; where X is selected from C, K, or Q.

2. The antibody of claim 1, wherein the antibody is selected from the group comprising IgG1, IgG2, IgG3, and IgG4.

3. The antibody of claim 2, wherein the constant region comprises one or more of the Ck, CH1 and CH3 domains of the IgG1 constant region.

4. The antibody of claim 1, wherein the antibody is selected from the group consisting of Fabs, bi specific antibody fragments (tandem scFv-Fc, scFv-Fc knobs-into-holes, scFv-Fc-scFv, F(ab′)2, Fab-scFv, (Fab′scFv)2, scDiabody-Fc, or scDiabody-CH3), IgG-based bispecific antibodies (Hybrid hybridoma, Knobs-into-holes with common light chain, Two-in-one IgG, Dual V domain IgG, IgG-scFv, scFv-IgG, IgG-V, V-IgG), minibody, tribi-minibody, nanobodies, and di-diabody.

5. The antibody of claim 1, wherein the antibody is selected from Abciximab; Rituximab; Basiliximab; Daclizumab; Palivizumab; Infliximab; Trastuzumab; Alemtuzumab; Adalimumab; Efalizumab; Cetuximab; Ibritumomab; Omalizumab; Bevacizumab; Ranibizumab; Golimumab; Canakinumab; Ustekinumab; Tocilizumab; Ofatumumab; Belimumab; Ipilimumab; Brentuximab; Pertuzumab; Raxibacumab; Vedolizumab; Ramucirumab; Obinutuzumab; Siltuximab; Secukinumab; Dinutuximab.

6. The antibody of claim 1, which lacks one or more Fc effector functions.

7. An immunoconjugate comprising the antibody of claim 1, a payload, and a linker joining the payload to the antibody.

8. The immunoconjugate of claim 7, wherein the linker is selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-Succinimidyl 4-(2-pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), N-Succinimidyl, (4-iodo-acetyl) aminobenzoate (SIAB),), SPDB, hydrazone, maleimidocaproyl and 6-maleimidocaproyl-valine-citrulline-p-aminobenyloxycarbonyl (MC-vc-PAB); or is a branched linker which comprises a peptide chain and is derived from o-hydroxy p-amino benzylic alcohol, wherein the peptide chain is connected to the phenyl ring via the p-amino group, the payload is connected to the phenyl ring via the benzylic alcohol moiety, and the antibody is connected to the phenyl ring via the o-hydroxy group.

9. The immunoconjugate of claim 7, wherein the payload is selected from the group consisting of 90Y, 131I, 67Cu, 177Lu, 213Bi, 211At, dolastatin, vedotin, monomethyl auristatin F(MMAF), monomethyl auristatin E (MMAE); maytansinoids including DM1 and DM4, duocarmycin, duocarmycin analogs, calicheamicin, pyrrolobenzodiazepines (PBD), centanamycin, irinotecan, and doxorubicin, alpha-amanitin, melatonin, membrane disrupting peptide, Pseudomonas exotoxin A, Diphtheria toxin, ricin, polyethylene glycol, hydroxyethyl starch, and a mannosyl residue.

10. A pharmaceutical composition comprising an antibody according to claim 1, and a pharmaceutically acceptable diluent, carrier or excipient.

11. A method for generating an immunoconjugate, the method comprising conjugating the antibody of claim 1 to a payload.

12. An isolated or recombinant polynucleotide encoding the antibody of claim 1.

13. A vector comprising the polynucleotide of claim 12.

14. A host cell comprising the vector of claim 13.

15. A method of producing an antibody comprising: (a) providing a culture medium comprising the host cell of claim 14; and (b) placing the culture medium in conditions under which the antibody is expressed, and optionally (c) isolating the antibody.

16. The antibody of claim 1, further comprising a mutation at one or more of heavy chain positions 160, 190, 168, 170, 176 or 200.

17. The antibody of claim 1, wherein the light chain is a kappa light chain.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows levels of expression of candidate antibodies in 25 ml CHOK1SV cultures.

(2) FIG. 2 shows results of SEC-HPLC analysis for % monomer of conjugated and unconjugated variant antibodies, and % aggregation and fragmentation for conjugated variants.

(3) FIG. 3 shows extent of biotin-maleimide conjugation to light chains of antibody variants. Open bars represent unconjugated product, striped bars represent products with a single conjugated payload, and solid bars represent products with two conjugated payloads.

(4) FIG. 4 shows extent of biotin-maleimide conjugation to heavy chains of antibody variants. Open bars represent unconjugated product, striped bars represent products with a single conjugated payload, and solid bars represent products with two conjugated payloads.

(5) FIG. 5 shows calculated drug antibody ratio for the antibody variants of FIGS. 3 and 4.

(6) FIG. 6 shows percentage biotin decrease over time for the subset of antibody variants.

(7) FIG. 7 shows calculated drug to antibody ratio of some of the selected variants conjugated to MMAE.

(8) FIG. 8 shows the decrease in cell viability for different cell lines that have been exposed to different concentrations of the selected ADC over a period of 72 h. PBS=phosphate buffered saline; vc-MMAE=valine-citrulline-MMAE.

DETAILED DESCRIPTION OF THE INVENTION

(9) The present inventors have developed a process for rational design of modified antibodies to allow selection of antibodies having desired properties for production of antibodies conjugated to payloads such as for example ADCs (ADC variants). The design process incorporates in silico and in vitro screening steps. Thus, the antibodies of the present invention share a number of properties, as will be seen.

(10) To incorporate residues for site specific conjugation, it was decided to replace native residues with another residue such as cysteine residues in selected positions of antibody structures. Candidate variants were analysed (in silico and in vitro) for desirable properties including titre and aggregation, and optionally immunogenicity (in silico only). As an initial proof of concept, Herceptin (trastuzumab) was chosen as a model antibody, and conjugation optimization and analysis carried out with biotin maleimide.

(11) Criteria for selecting mutation sites included: Residues to be mutated to cysteine (cys) must have similar physicochemical properties or be a small non-hydrophobic non-charged residue (ser, val, thr, ala) Residues amenable to be mutated to cysteine must be in constant regions of either light chain (C.sub.K, Cλ,) or heavy chain (CH1, CH2 or CH3) of an antibody or a fragment thereof. In cases where a modified antibody or scaffold is used, introduced cys should be at a distance >5 Å from any target-binding interface or domain to minimise risk of interfering with biological activity of the molecule. Mutations to cys should not create intra-chain hydrogen bonds leading to the alteration of the local environment and the properties of the protein Mutations to cys must not be placed in the interfaces between chains or domains of the antibody (or scaffold). As a general rule modifications should be at a distance >5 Å from residues involved in either chain-chain or domain-domain interfaces. Mutations to cys should be at a distance >5 Å from any antibody native cys and should not interfere with the Fc glycosylation site (i.e. should be placed at a distance >5 Å from residue Asn295 where glycosylation occurs) Mutations to cys should not increase the chemical degradation risk/should not introduce undesired post translational modifications

(12) Screening of the trastuzumab sequence was then carried out to identify suitable sites for mutation to another residue such as e.g. cysteine.

(13) The unmodified light chain sequence is:

(14) TABLE-US-00003 (SEQ ID NO: 1) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYS ASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQ GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

(15) The unmodified heavy chain sequence is:

(16) TABLE-US-00004 (SEQ ID NO: 2) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR IYPINGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYQSRWG GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFFAVEQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLEPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(17) Ser, Thr, Val, and Ala residues in CH1, CH3, and CL were explored. This gave a number of candidates: Light: SER171, VAL191, SER208, SER182, THR180, THR206, ALA184, SER203, SER202 Heavy: VAL170, VAL176, THR190, SER445, SER443, SER139, SER160, SER447, THR200, SER168.

(18) These mutations were then analysed for desirable properties. Solvent accessibility surface modelling was carried out in silico. Discovery Studio (Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.0, San Diego: Accelrys Software Inc., 2013.) was used to calculate the Side Chain Solvent Accessibility Surface of the chosen residues. Solvent accessibility should be greater than 15% (>15%), or greater than 17% (>17%) to facilitate ‘conjugability’ of the molecule. The percentage side chain solvent accessibility surface is calculated as 100 times the side chain solvent accessibility divided by the side chain solvent accessibility of the fully exposed amino acid residue calculated using the extended Ala-X-Ala tripeptide, where X is the residue of interest. Side chains with solvent accessibility ratios of equal to or less than 15% (<=15%) or equal to or less than 17% (<=17%) are considered buried and not taken into account. The results of the SAS modelling are shown below:

(19) TABLE-US-00005 Variant Variant LC % SAS HC % SAS SER171 5.45 VAL170 17.74 VAL191 48.069 VAL176 25.179 SER208 48.585 THR190 27.548 SER182 67.216 SER445 33.961 THR180 67.518 SER443 46.173 THR206 75.247 SER139 53.401 ALA184 116.94 SER160 61.951 SER203 122.689 SER447 72.323 SER202 136.452 THR200 91.354 SER168 133.127

(20) Aggregation propensity modelling was also carried out in silico. Aggregation propensity should not be significantly increased by the introduction of the intended engineered Cys of any of these (potential) ADC variants. This propensity will be calculated based on a Z score comparison of the reference molecule and any of the (potential) ADC variants described above to the distribution of values for a reference set of the smallest functional domain of the antibody or protein where the mutation to Cys is introduced. A mean and standard deviation is determined for the reference set. The Z-score is then calculated by subtracting the reference mean from the target proteins score and dividing by the standard deviation. The result is a zero (0) centred score where positive values indicated that the target is more aggregation prone (in this case) than the mean. Targets with a Z-score within (−1, 1) are within the standard deviation of the score within the reference set. The AggreSolve™ in silico platform (Lonza, Basel, Switzerland) comprises a collection of algorithms which, based on sequence and structural parameters, can calculate predictors that reflect the aggregation propensity of a given polypeptide. Such predictors reflect global and local (residue-specific) aggregation propensities as well as local flexibility and stability.

(21) TABLE-US-00006 Difference Antibody Name Z-score (Variant − WT) Trastuzumab Heavy Chain (H) 0.36 H:S160C 0.02 −0.34 H:T190C 0.36 0.00 H:S443C 0.19 −0.17 H:S447C 0.19 −0.17 Trastuzumab Light Chain (L) 3.09 L:T180C 2.85 −0.24 L:T206C 3.10 0.01 Trastuzumab Heavy Chain constant 0.77 domain 1 (CH1) CH1:S160C 0.17 −0.60 CH1:T190C 0.80 0.04 Trastuzumab Heavy Chain constant −0.07 domain 3 (CH3) CH3:S443C −0.33 −0.26 CH3:S447C −0.36 −0.29 Trastuzumab Light Chain constant 1.95 domain (CL) CL:T180C 1.66 −0.29 CL:T206C 1.98 0.03

(22) The AggreSolve Z-score has been calculated for the full length Trastuzumab heavy and light chain, as well as for the CH1, CH3, and CL domains in which the ADC substitutions are located (the minimal functional domains).

(23) The boundaries for the CH1, CH3 and CL domains are as per the IMGT definition in M. P. Lefranc, C. Pommie, Q. Kaas, E. Duprat, N. Bosc, D. Guiraudou, C. Jean, M. Ruiz, I. Da Piedade, M. Rouard, E. Foulquier, V. Thouvenin, and G. Lefranc. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains. Developmental and comparative immunology 29 (3), 2005.

(24) Following these in silico selection steps, in vitro tests were carried out on the variants to determine protein yield (in vitro), aggregation/fragmentation, and binding kinetics. Protein yield, as estimated by product titre in supernatant and after protein A purification must be at least 70% or higher of the parental molecule The percentage of monomer lost after conjugation measured through Size Exclusion Chromatography HPLC) is preferably ≤35%, more preferably ≤5%, ≤10%, ≤15%, ≤20%, ≤25%, or ≤30%. Percentage aggregation of the conjugated molecule is preferably <5%, <10%, <15%, or <20%. Percentage fragmentation of the conjugated antibody is preferably <5%, <10%, <15%, <20%, <25%, <30%, <32%, <35% or <40%. The Constant of Dissociation (KD) of the conjugated variants must be equal to or less than 2 (≤2) orders of magnitude than the reference standard KD of an unmutated or parent antibody. For the antibodies described herein the reference standard is herceptin (trastuzumab), although it will be appreciated that where a different parent antibody is used, then that parent may be used as a reference standard.

(25) Systems for cloning and expression of antibodies in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common, preferred bacterial host for small immunoglobulin molecules is E. coli. The expression of immunoglobulins, such as antibodies and antibody fragments, in prokaryotic cells such as E. coli is well established in the art. Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a immunoglobulin. Immunoglobulins, such as antibodies and antibody fragments, may also be expressed in cell-free systems.

(26) Suitable vectors for the expression of immunoglobulins can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. Nucleic acid encoding a variant immunoglobulin or a CH1, VH and/or VL domain thereof may be contained in a host cell.

(27) Variant antibodies were generated as described for example in WO2011021009A1. In detail: DNA encoding the antibody variants as described herein were chemically synthesized and cloned into a suitable mammalian expression vector. For transient expression experiments heavy and light chain were cloned into separate expression vectors. For generation of cell lines stably expressing a variant antibody heavy and light chains were cloned into one single expression vector. Each expression vector comprises a DNA encoding a signal sequence upstream of the heavy chain and the light chain coding regions to enable secretion of the heavy and light chain from the mammalian cells.

(28) For transient expression, CHOK1SV cells were transfected using for example Lipofectamine with the expression vectors encoding the variants as described herein.

(29) For example in case of variants comprising at least one mutation in the light chain, an expression vector comprising said mutation(s) was co-transfected with a vector encoding the unmodified heavy chain; in case of variants comprising at least one mutation in the heavy chain, an expression vector comprising said mutation(s) was co-transfected with a vector encoding the unmodified light chain. 72 h post-transfection, supernatants were harvested form the transfected cells, centrifuged and stored at 4° C. prior to purification.

(30) For Large scale production CHOK1SV cells are transfected as described above with a single vector comprising modified or unmodified light and heavy chain. Either pools of stably transfected cell are used for further experiments or a clonal selection is performed. Supernatants of such stable transfected cells expressing a variant of the present invention was harvested and stored at 4° C. prior to purification.

(31) Cell culture supernatants were Protein A purified using HiTrap columns (GE) and stored at 4° C. prior to concentration and buffer exchange. Samples were concentrated by centrifugation at 2000 g 15-20 min. Material was buffer exchanged 4-5 times using formulation buffer (50 mM Phosphate, 100 mM NaCl, pH7.4). Once buffer exchanged, samples were diluted in formulation buffer to an appropriate working concentration.

(32) Protein Yield Assessment (In Vitro)

(33) The antibody or antibody variant yield is estimated by product titre in supernatant and after protein A purification (e.g. through sandwich ELISA, with absorbance at 280 nm, or via HPLC protein A quantification).

(34) FIG. 1 shows levels of expression of each candidate antibody in 25 ml CHOK1SV cultures. All variants show similar levels of expression.

(35) Conjugation

(36) Conjugation was carried out with biotin-maleimide conjugation to free thiol groups by standard techniques Junutula J R et al, Nature Biotechnology 2008, 8, 925-932; Jeffrey S C et al, Bioconjugate Chem. 2013, 24, 1256-1263.

(37) For conjugation to a toxin engineered antibodies are e.g. reduced with a tris(2-carboxyethyl)phosphine (12.5 eq.) for 2 h at 35° C. and pH 7.7. The mixture is buffer exchanged into 50 mM Tris, 5 mM EDTA, pH 7.7. Dehydroascorbic acid (15 eq.) is added and the oxidation reaction allowed to proceed for 3 h at 24° C. N,N-dimethylacetamide is added to reach a concentration of typically between 1 and 5%. Maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethylauristatin E (5 eq.) is added and the conjugation reaction allowed to proceed for 1 h at 22° C. The reaction is quenched by addition of N-acetyl-cysteine (5 eq.). Following 0.5 h incubation at 22° C., the mixture is buffered exchanged into 1×PBS.

(38) Aggregation Propensity/Fragmentation Assessment (In Vitro)

(39) Before and after conjugation the percentage of monomer lost due to antibody aggregation and/or fragmentation was measured quantitatively using Size Exclusion Chromatography HPLC (SEC-HPLC) and qualitatively using SDS PAGE. For the latter, each variant antibody was treated with beta mercaptoethanol, or given no treatment, and size fractionated on a SDS PAGE. There was no apparent aggregation or fragmentation of the variants visible.

(40) Results from SEC-HPLC analysis of conjugated and unconjugated samples are shown in FIG. 2, using samples at 1 mg/ml on a Zorbax-250GF column. Surprisingly some of the antibodies of the invention such as for example HC S139C, HC V170C, HC S160C, HC T200C, LC V191C, LC T2060, and LC T1800 showed less monomer lost, and/or decreased amount of antibody aggregation or fragmentation compared with the parent/unmutated antibody.

(41) Binding Kinetics Assessment (In Vitro)

(42) Binding kinetics of the variants were also analysed using a quartz crystal microbalance. ERB2/HER2 Fc chimaera were immobilized to carboxyl chip, and three different concentrations of each variant (conjugated and not conjugated) were tested. The table below summarises the Kd for each variant:

(43) TABLE-US-00007 K.sub.D (nM) Reference Not Conjugated Conjugated Herceptin 3.0 1.63 Variant Not Conjugated Conjugated LCHerS208C — — HCHerS443C 4.36 31.13 LCHerS202C 12.22 0.72 HCHerT200C 3.52 12.91 HCHerV170C 3.43 2.90 HCHerS447C 3.17 1.34 LCHerV191C 4.94 3.56 HCHerS445C 1.27 1.74 HCHerS168C 18.11 1.55 HCHerT190C 1.26 2.28 HCHerS139C 3.56 2.44 LCHerT206C 9.41 19.91 LCHerT180C 5.19 1.46 HCHerS160C 9.76 1.50 LCHerS182C 5.28 0.008 LCHerA184C 1.25 1.04 LCHerS203C — —
Drug to Antibody Ratio (DAR) Assessment (In Vitro)

(44) Finally, the DAR was determined for each of the variants, by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS or LC-ESI-MS). The DAR values for the different variants must be >1.7 and <2.2, as there are two site specific conjugation sites per antibody.

(45) For determining the DAR samples at 1 mg/ml were treated with PNGaseF. Reduced and not-reduced samples were analysed, by RP chromatography, electrospray, and mass spectrometry.

(46) The extent of biotin-maleimide conjugation to light and heavy chains of the variants is shown in FIGS. 3 and 4 respectively, with the calculated DAR for each variant being shown in FIG. 5.

(47) Conjugation and De-Conjugation Assessment (In Vitro)

(48) When stability of the ADC is analysed in vitro, the percentage average loss of conjugated molecule over a period of 8 days should be <39%.

(49) Six preferred variants were selected, and each of the preferred six variants was then further analysed. Conjugate stability (levels of deconjugation) was determined for four different concentrations (150 ng/ml; 300 ng/ml; 1000 ng/ml and 2000 ng/ml) of each of the final variants in human serum at 37 deg C. for 8 days. Samples were taken on days 0, 2, 4, and 8, and analysed by ELISA.

(50) The percentage biotin decrease over 8 days is shown in FIG. 6.

(51) Each of the final six variants was then ranked for desirable characteristics (purity, DAR, deconjugation, and positive environment), and given a score from 6 (=best) to 1 (=worst). The scores were then totaled, to give an overall score from 4 to 24. This gives an indication of the desirability of each antibody for further development. The scores are shown in the table below.

(52) TABLE-US-00008 % Decon- Positive Purity DAR jugation Environment Total HCHerS443C 60.83 1 2.00 6 29 6 LYS 6 19 HCHerS447C 66.94 2 2.09 4 40 1 2 9 HCHerT1900 77.19 3 1.77 2 37 3 2 10 LCHerT206C 82.7 5 2.13 3 40 1 LYS 6 15 LCHerT180C 84.58 6 1.76 1 32 5 2 14 HCHerS160C 81.03 4 1.96 5 32 5 2 16

(53) Although the antibody variants can be ranked in this way, as each of the final six has been through the initial selection process, they can all be said to have desirable characteristics for development as an ADC. In particular, not every antibody will make it through subsequent drug development processes and in vivo testing, so it is beneficial to be able to generate a selection of candidates. Furthermore, other variants not selected for the final six, such as the remaining variants disclosed herein, may also have beneficial properties and so may be considered useful for further investigation.

(54) The six final variants were: four heavy chain (S160C, T1900, S443C, S447C), and two light chain (T180C, or T206C) variants. As a result of the sequential method of selection all final variants can be expected to share a number of specific properties (or design criteria): Stability; low aggregation; low chemical degradation risk; low undesired post translational modifications; structural stability preserved; productivity; suitability for being conjugated; and biological activity.

(55) The values for each tested variant are shown in the table below; the six final selected variants are highlighted.

(56) TABLE-US-00009 SEC K.sub.D (nM) Prot A HPLC % NOT LC MS Light Chain LC MS Heavy Chain HPLC Monomer CON- CON- Un- +1 Con- +2 Con- Un- +1 Con- +2 Con- +3 Con- mg/L conj, Ab JUGATED JUGATED modified jugate jugates modified jugate jugates jugates DAR Herceptin 55.13 78.53 3.02 1.63 100%  0% 0% 100%  0%  0% 0% 0.00% LCHerS208C 48.11 54.06 — — — — — — — — — — HCHerS443C 54.96 60.83 4.36 31.13  89% 11% 0%  11% 89%  0% 0% 2.00% LCHerS202C 55.00 69.00 12.22 0.72  32% 60% 7%  95%  5%  0% 0% 1.60% HCHerT200C 58.83 94.94 3.52 12.91 100%  0% 0% 100%  0%  0% 0% 0.00% HCHerV170C 55.42 93.54 3.43 2.90 100%  0% 0% 100%  0%  0% 0% 0.00% HCHerS447C 49.11 66.94 3.17 1.34  81% 19% 0%  15% 85%  0% 0% 2.09% LCHerV191C 56.37 95.73 4.94 3.56 100%  0% 0% 100%  0%  0% 0% 0.00% HCHerS445C 57.93 77.22 1.27 1.74  78% 22% 0%  22% 35% 34% 8% 3.01% HCHerS168C 57.68 78.01 18.11 1.55  93%  7% 0%  39% 61%  0% 0% 1.36% HCHerT190C 46.22 77.19 1.26 2.28  94%  6% 0%  18% 82%  0% 0% 1.77% HCHerS139C 60.58 80.27 3.56 2.44  84% 16% 0%  22% 52% 22% 4% 2.47% LCHerT206C 53.83 82.70 9.41 19.91  4% 88% 8%  97%  3%  0% 0% 2.13% LCHerT180C 46.87 84.58 5.19 1.46  16% 81% 4% 100%  0%  0% 0% 1.76% HCHerS160C 55.47 81.03 9.76 1.50  95%  5% 0%  7% 93%  0% 0% 1.96% LCHerS182C 51.18 73.15 5.28 8 × 10 − 3  38% 62% 0% 100%  0%  0% 0% 1.25% LCHerA184C 60.11 80.20 1.25 1.04  86% 14% 0% 100%  0%  0% 0% 0.28% LCHerS203C 53.61 58.22 — — — — — — — — — —

(57) Subsequently the final six variants were conjugated to Monomethyl Auristatin E (MMAE) by standard techniques. The conjugation method follows broadly methods described above. The DAR for the selected variants was determined as described above. An example of the results is shown in FIG. 7. Cond 1-3 represent minor variants in the conjugation procedure with parameters varied to try to optimise the DAR; the reduction time and temperature for antibodies prior to conjugation were varied in each of conditions 1-3: Condition 1: reduction at 35° C. for 2 h (as described above) Condition 2: reduction at 25° C. for 2 h Condition 3: reduction at 35° C. for 1 h.

(58) A double mutant (DM) combining LC T180C and HC S160C was also tested to determine aggregation propensity and DAR data, using the same techniques as described above. The results are shown in the following tables:

(59) DM Aggregation Data from the Transient Transfections

(60) using size-exclusion chromatography SEC

(61) TABLE-US-00010 Relative % by SEC Species Before conjugation After conjugation Purity, main peak 95.4 96.1 High molecular weight forms  3.0  3.0 Low molecular weight forms  1.6  0.9
DM DAR Data from the Transient Transfections using PLRP HPLC or ESI-MS methods

(62) TABLE-US-00011 Variant PLRP Intact mass DM 3.77 3.97
In Vitro Toxicity Tests

(63) After MMAE conjugation the ADC variants were tested for in vitro cytotoxicity. The analysis was carried out by standard techniques (Andreotti, P. E. et al. Cancer Res 1995.55, 5276-82; Gerhardt, R. T. et al. Am. J. Obstet. Gynecol 1991 165, 245-55). The cells chosen for the assay were based on Neve R. M. et al. Cancer Cell 2006 10, 515-527.

(64) Assay Schematics:

(65) Day 1: Seed three 96-well plates each of SKBR3 cells (5 k/well) in media (McCoy5A+10% FBS+1×Pen/Strep), BT474 cells (8 k/well) in media (DMEM/F12+10% FBS+1×Pen/Strep), and MCF7 cells (4 k/well) in media (RPMI+10% FBS+1×Pen/Strep). Incubate in 37° C. humidified CO2 incubator for 18 hrs.

(66) Day 2: Prepare ADC variants sample dilutions. Make the initial 667 nM working stocks of these samples in RPMI media with 10% FBS. Then prepare ⅓ serial dilution from 667 nM to 11 pM in media. Add 5 ul of the dilution into each well of ˜100 ul cells. Final sample concentrations range from 33.3 nM to 0.56 pM (as ⅓ serial dilutions). Incubate at 37° C. in a humidified CO2 incubator for 72 hrs.

(67) Day 4: Evaluate the plates under a microscope

(68) Day 5: Determine cell viability using Cell-Titer Glo reagent: Aspirate the media from the 96-well plate. Add 100 ul of Cell-Titer Glo reagent (Promega Inc.) in each well. Incubate at room temperature for 10 min. Determine luminescence using Tecan Ultra plate reader. Analyze and plot data either as Percent Viability vs. Concentration (nM), or as random luminesce.

(69) An example of the results is shown in FIG. 8. As can be seen in FIG. 8, ADC variants HC S443C and LC T180C reduce the viability of SKBR3 cells and BT474 cells by 50% at very low concentrations, whereas these ADC variants do not show an effect over 72 hours in less responsive cells like MCF7.

(70) The full sequences of the variant chain of each of the variants described herein are shown below. These show only the variant chain; the other chain will be the same as the unmodified trastuzumab sequence (that is, SEQ ID No 1 (LC) or 2 (HC)).

(71) TABLE-US-00012 Heavy chains: >HCherS139C (SEQ ID No 3) EVQLVESGGGLVQPGGSLRLSCAASGENIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTCGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK >HCherS160C (SEQ ID No 4) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVCWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK >HCherS168C (SEQ ID No 5) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTCGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK >HCherV170C (SEQ ID No 6) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGCHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMH EALHNHYTQKSLSLSPGK >HCherV176C (SEQ ID No 7) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPACLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK >HCherT190C (SEQ ID No 8) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVCVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK >HCherT200C (SEQ ID No 9) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQCYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK >HCherS443C (SEQ ID No 10) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKCLSLSPGK >HCherS445C (SEQ ID No 11) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLCLSPGK >HCherS447C (SEQ ID No 12) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWV ARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYC SRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLCPGK Light chains: >LCherS171C (SEQ ID No 13) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDCTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC >LCherT180C (SEQ ID No 14) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLCLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC >LCherS182C (SEQ ID No 15) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TEGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLCKADYEKEKVY ACEVTHQGLSSPVTKSFNRGEC >LCherA184C (SEQ ID No 16) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKCDYEKHKVY ACEVTHQGLSSPVTKSFNRGEC >LCherV191C (SEQ ID No 17) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKCY ACEVTHQGLSSPVTKSFNRGEC >LCherS202C (SEQ ID No 18) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLCSPVTKSFNRGEC >LCherS203C (SEQ ID No 19) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSCPVTKSFNRGEC >LCherT206C (SEQ ID No 20) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVCKSFNRGEC >LCherS208C (SEQ ID No 21) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKCFNRGEC

(72) It will be appreciated that a similar selection and screening process may be used to develop other variant antibodies, not only those based on trastuzumab, and further that it may be expected that variants of these other antibodies having the same constant region mutations as identified herein would also be expected to have similar desirable properties.