Processes for Constructing Homogeneous Antibody Drug Conjugates

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Processes for Constructing Homogeneous Antibody Drug Conjugates David Young Jackson Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00067 • Publication Date (Web): 14 Apr 2016 Downloaded from http://pubs.acs.org on April 18, 2016

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Organic Process Research & Development

Processes for Constructing Homogeneous Antibody Drug Conjugates

David Y. Jackson †, *

†

Igenica Biotherapeutics, 863A Mitten Road, Suite 100B, Burlingame, CA 94010, USA

* Corresponding Author: David Y. Jackson, Ph.D. Igenica Biotherapeutics 863A Mitten Road, Suite 100B Burlingame, CA 94010, USA Email: [email protected] Cell: 650-339-3948

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T.O.C. Graphic

S

S

TAG

Engineered AAs

TAG

Enzyme Mediated

Linker-Based


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ABSTRACT: Antibody drug conjugates (ADCs) are synthesized by conjugating a cytotoxic drug or „payload‟ to a monoclonal antibody. The payloads are conjugated using amino or sulfhydryl specific linkers that react with lysines or cysteines on the antibody surface. A typical antibody contains over sixty lysines and up to twelve cysteines as potential conjugation sites. The desired DAR (drugs/antibody ratio) depends on a number of different factors and ranges from two to eight drugs/antibody. The discrepancy between the number of potential conjugation sites and the desired DAR, combined with use of conventional conjugation methods that are not site-specific, results in heterogeneous ADCs that vary in both DAR and conjugation sites. Heterogeneous ADCs contain significant fractions with suboptimal DARs that are known to possess undesired pharmacological properties. As a result, new methods for synthesizing homogeneous ADCs have been developed in order to increase their potential as therapeutic agents. This article will review recently reported processes for preparing ADCs with improved homogeneity. The advantages and potential limitations of each process are discussed, with emphasis on efficiency, quality and in vivo efficacy relative to similar heterogeneous ADCs. Keywords: antibody drug conjugate, ADC, site-specific, homogeneous, linker, payload  INTRODUCTION Antibody drug conjugates (ADCs) are a rapidly growing class of targeted therapeutic agents for treatment of cancer. 1-8 Although the number of ADCs in clinical trials has steadily increased since 2005, many have failed to reach the later stages of clinical development, one has been withdrawn from the market (Mylotarg in 2002), and only two (Adcetris and Kadcyla) are currently approved by the FDA for cancer indications (Figure 1A). 9-11 Thus far, the approval rate for ADCs has not met early expectations and is lagging behind other antibody-based therapeutics. Based on the number of approved ADCs versus those that have failed to progress into later stage clinical trials, the success rate is reminiscent of that for small molecule drugs. The reasons for the clinical failures of ADCs are often not known or they are still under investigation. More commonly, when the reasons for clinical failure are clear, the information is not made available to the public domain. Emerging preclinical data suggests that heterogeneity, a property shared by most ADCs currently in clinical development (Table 1), may ultimately limit their potential as therapeutic agents.12, 13

Table 1. Examples of heterogeneous ADCs currently in clinical trials for cancer indications. (Source: www.clinicaltrials.gov)


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ADC

Sponsor

Indications

Status

Payload

Linked to

Target

Adcetris

Seattle Genetics

HL & ALCL

Approved

MMAE

Cysteine

CD30

Kadcyla

Genentech/Roche

Breast Cancer

Approved

DM1

Lysine

Her2

Inotuzumab ozogamicin

Pfizer

NHL & ALL

Phase III

Calicheamicin Lysine

CD22

Lorvotuzumab mertansine

Immunogen

SCLC

Phase II

DM1

Lysine

CD56

Glembatumumab vedotin

Celldex

BC, Melanoma Phase II

MMAE

Cysteine

GPNMB

PSMA- ADC

Progenics

Prostate

Phase II

MMAE

Cysteine

FOLH1

SAR-3419

Sanofi

DLBCL, ALL

Phase II

DM4

Lysine

CD19

ABT-414

Abbvie

Glioblastoma

Phase II

MMAE

Cysteine

EGFR

BT-062

Biotest

Mult. Myeloma Phase II

DM4

Lysine

CD138

HLL1-Dox

Immunomedics

CLL, MM, NHL Phase II

Doxorubicin

Cysteine

CD74

Immu-130

Immunomedics

CRC

Phase II

SN-38

Cysteine

CEACAM5

Immu-132

Immunomedics

Solid tumors

Phase II

SN-38

Cysteine

EGP1

SYD985

Synthon

Breast Cancer

Phase II

Duocarmycin

Cysteine

Her2

SAR-3419

Sanofi

DLBCL, ALL

Phase II

DM4

Lysine

CD19

IMGN853

ImmunoGen

Solid tumors

Phase I

DM4

Lysine

FOLR1

IMGN529

ImmunoGen

BCL,CLL, NHL Phase I

DM1

Lysine

CD37

ASG-22M6E

Astellas

Solid tumors

Phase I

MMAE

Cysteine

Nectin-4

AGS-16M8F

Astellas

RCC

Phase I

MMAF

Cysteine

AGS16

AMG 172

Amgen

RCC

Phase I

DM1

Lysine

CD27L

AMG 595

Amgen

Glioblastoma

Phase I

DM1

Lysine

EGFR8

BAY94-9343

Bayer

Solid tumors

Phase I

DM4

Lysine

Mesothelin

ADCs are composed of a cytotoxic drug or „payload' conjugated to a tumor selective monoclonal antibody. The heterogeneity of conventional ADCs arises from the synthetic processes currently used for conjugation.

14

Payloads are typically conjugated to the antibody using amino or thiol specific linkers

that react with lysines or cysteines on the antibody surface.

15

A typical antibody contains more than

fifty lysines and up to twelve cysteines as potential conjugation sites (Figure 1B). 16 The optimal DAR (drugs/antibody ratio) for most ADCs however, ranges from 2 to 8 drugs/antibody and is dependent upon a variety of different factors. The discrepancy between the number of potential conjugation sites and the desired DAR, combined with the use of conjugation methods that are not site-specific, result in heterogeneous ADCs that vary in both DAR and conjugation sites. Consequently, conventional heterogeneous ADCs often contain significant amounts of unconjugated antibody in addition to fractions with sub-optimal DARs. Unconjugated antibodies can compete for antigen binding and inhibit ADC


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activity, while fractions with sub-optimal DARs are frequently prone to aggregation, poor solubility and/or instability that ultimately result in a poor therapeutic window. 17, 18

The relative degree of ADC heterogeneity depends on the methods used for conjugation. For example, KadcylaTM, an ADC approved in 2013 for breast cancer, is synthesized using a two-step process in which the linker and payload are conjugated in separate steps (Scheme 1A).

19-21

The linker

contains an amino-specific NHS ester that reacts with antibody lysines in the first step, and a thiolspecific maleimide group that reacts with a maytansinoid payload in the second step. The process affords a highly heterogeneous mixture of ADC molecules containing from 0 to 10 payloads/antibody with an average DAR of 3.5 drugs/antibody.

22, 23

Additional heterogeneity arises due to distribution of

the payloads across dozens of potential conjugation sites. As a result, Kadcyla contains hundreds of different ADC molecules, each with its own unique pharmacological properties. 24 Conjugation of payloads to antibodies through inter-chain cysteines reduces ADC heterogeneity relative to lysine conjugation because there are fewer potential conjugation sites. AdcetrisTM, an ADC approved in 2011 for treatment of Hodgkin‟s lymphoma, is an example of a cysteine conjugated ADC.2527

The process for cysteine conjugation involves partial reduction of four antibody inter-chain disulfide

bonds to generate up to eight reactive thiol groups. The partially reduced antibody is subsequently conjugated to a payload containing a thiol-specific maleimide linker. The payload used for Adcetris is monomethyl auristatin E (MMAE), and contains a protease cleavable maleimide linker, (Scheme 1B). Although Adcetris is less heterogeneous than Kadcyla, it is composed of dozens of different ADC molecules containing 0 to 8 payloads with an average DAR of 3.6 drugs/antibody. 28 Like most cysteine ACS Paragon Plus Environment

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conjugated ADCs, Adcetris has a reduced half-life in vivo compared to the parent antibody, cAC10. The diminished half-life has been attributed to rapid clearance of high DAR species (> 4 drugs/antibody) and to partial loss of inter-chain disulfide bonds during the conjugation process.29, 30 Although different processes for lysine and cysteine conjugation are used to synthesize Adcetris and Kadcyla, both ADCs contain thio-succinimide bonds between the payload and the antibody, which originate from the use of maleimide linkers in the conjugation processes. Kadcyla contains a thiosuccinimide between the linker and the payload (Scheme 1A), while Adcetris contains a thiosuccinimide bond between the linker and the antibody (Scheme 1B). Thio-succinimide groups are known to undergo undesired side reactions such as elimination or thiol exchange that can result in premature release of the payloads from the ADC and lead to reduced potency and/or increased systemic toxicity.31, 32

Despite the known limitations of conventional heterogeneous ADCs, most ADCs currently in clinical development utilize similar conjugation methods to those described in scheme 1. As a result, they are likely to possess similar pharmacological properties to Adcetris and Kadcyla, in addition to other less successful ADCs that may have performed poorly in clinical trials. In order to improve the pharmacological properties of current and future ADCs, new site-specific conjugation processes for synthesizing homogeneous ADCs are now being developed. 33-36


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Site-specific conjugation processes for constructing homogeneous ADCs can be divided into three different categories. Two are focused on antibody modification (engineered amino acids and enzyme mediated), while the third category is focused on linker modification. The categories can be sub-divided further based on the specific processes that are used (Table 2). Examples from each process were selected based on availability of sufficient preclinical data to enable comparison with similar conventional heterogeneous ADCs. Homogeneous ADCs derived from these processes have only just begun to enter clinical trials. Whether they will outperform their heterogeneous counterparts in clinical trials remains uncertain, but preclinical data suggests that homogeneous ADCs are likely to dominate future clinical trials and will lead to improved clinical results.

Enzyme Mediated

Engineered A.A.s

Table 2. Summary of different processes for constructing homogeneous ADCs.

LInker-Based

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Institution

Approach

Conjugation site

Linker type

Payload(s)

DAR

References

Seattle Genetics

Engineered Cysteines

HC (S239)

Maleimide

MMAE, PBD

2

42, 50, 51

Genentech


HC(A114),LC(V205)

Maleimide

MMAE, DM1

2 or 4

43-46, 48

ETH


N or C terminus

Aldehyde

Cemadotin

2

47

AmBrx /WuXi

Eng. p-acetyl Phe

HC (S115)

Alkoxyamine

MMAD

2

55

Sutro/Cellgene

Eng. p-azido-Phe

HC (S136)

Alkyne

MMAF

2

56-57

Allozyne/Medimmune

Eng. azido-Lys

HC (K274)

Alkyne

AF or PBD

2

58

Pfizer/Rinat

Transglutaminase (mTG)

LLQGA tag

1 0 Amine

MMAD

2

59 - 61

Innate Pharma

Transglutaminase (BTG)

HC (Q295,Q297)

1 Amine

MMAE

4

62 - 64

Catalent/Redwood Biosciences

Formylglycine generating enzyme (FGE)

CXPXR tag

Hydrazone

DM1

2

65 - 67

NBE Therapeutics

Sortase A

LPETG tag

1 0 Amine

DM1

2 or 4

68

Sanofi/Genzyme

Glycosyl transferase

HC(N297)-CHO

Alkoxyamine

MMAE

1 or 2

69

SynAffix

Endoglycosidase

HC(N297)-AzidoLys

Alkyne

DM1

2

70

Seattle Genetics

Hydrophylic linkers

Interchain Cysteines

Maleimide

Auristatin T

8

72

Polytherics

Disulfide Bridging


Bis-sulphone

MMAE

4

74 - 76

University College of London (UCL)

Next Generation Maleimides (NGMs)


Dithiophenylmaleimide Dibromopyridazinedione

Doxirubicin

4

77-89

Igenica

Interchain Cross-linking


Dibromomaleimide

MMAF

4

90, 91

0

 Engineered Amino Acid Approaches Early attempts to construct homogeneous ADCs were performed by reduction of interchain disulfide bonds followed by conjugation of payloads to all eight interchain cysteines. 37, 38 The process resulted in a loss of four interchain disulfide bonds and frequently resulted in ADC aggregation, instability, and/or poor solubility due to hydrophobic properties of the payloads that ACS Paragon Plus Environment

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were available at that time. These fully loaded ADCs containing eight drugs/antibody also demonstrated poor pharmacokinetic properties and offered no significant advantages over analogous heterogeneous ADCs with lower DARs. 17, 30, 39 The conjugation methods currently used for preparing conventional heterogeneous ADCs such as Adcetris or Kadcyla were developed in order to overcome the potential liabilities associated with fully loaded ADCs. Heterogeneity was considered an acceptable penalty for the benefits gained by lowering DARs. It has now become apparent that new methods for synthesizing homogeneous ADCs are necessary for ADCs to reach their full therapeutic potential. Most new methods for synthesis of homogeneous ADCs require recombinant engineering in order to introduce unique functional groups into the antibody for site-specific conjugation. In early examples of site-specific conjugation, Rader and coworkers incorporated selenocysteine into antibodies to obtain ADCs with one or two drugs per antibody.

40, 41

More recently, several different engineered amino acid

approaches have been used successfully to generate homogeneous ADCs with two, four or eight drugs per antibody. In most cases, the engineered ADCs have outperformed similar heterogeneous ADCs in vitro and in vivo, yet there are potential limitations that should be considered prior to clinical development. For instance, recombinant methods for antibody re-engineering are not applicable to existing „off-the-shelf‟ antibodies, which might be desirable in some cases. Other potential challenges for recombinant approaches include identification of optimal conjugation sites, possible immunogenicity and use of antibody expression systems which have not yet been clinically validated. Whether the benefits of ADC homogeneity will outweigh the additional time and cost associated with developing these methods is still unclear, but significant progress has been made toward producing homogeneous ADCs with improved pharmacological properties. Engineered Cysteines The first examples in which recombinant antibody engineering was used to improve ADC homogeneity involved two opposite strategies, removal or addition of cysteine residues. Carter and coworkers systematically removed interchain cysteines by replacement with serine in cAC10, the antiCD30 antibody used in Adcetris.

42

The remaining cysteines were then conjugated to the well-known

auristatin payload (MC-vc-Pab-MMAE) to yield homogeneous ADCs containing two or four drugs/antibody (Scheme 2A). The resulting ADCs were found to have comparable pharmacological properties to analogous heterogeneous ADCs. This led the authors to conclude that improved homogeneity had a minimal effect on the therapeutic index of ADCs; however, the loss of interchain


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disulfides in the engineered ADCs may have masked potential gains derived from improved homogeneity. An alternative approach by Junutula and coworkers, led to a different conclusion. Cysteine mutations were introduced into position 114 on the heavy chain of an anti-MUC16 antibody, 3A5.43 The mutations provided two unique unpaired thiol groups suitable for conjugation to payloads containing conventional maleimide linkers. The process afforded ADCs that contained predominantly two drugs/antibody, however, additional reduction and oxidation steps were required to obtain mutant antibodies in a form suitable for conjugation (Scheme 2B). The thio-mAb ADC (aka TDC) demonstrated comparable efficacy to a conventional heterogeneous ADC, yet the relative toxicity of the TDC was significantly reduced for an improved therapeutic index. In a subsequent study, Boswell and coworkers used a similar approach to construct anti-STEAP1 ADCs and obtained comparable results.44 The engineered cysteine approach was later applied to alternative payloads via site-specific conjugation of engineered trastuzumab to a maytansine payload (DM1).

45

The DM1 payload is

analogous to that used in Kadcyla, a heterogeneous trastuzumab ADC approved in 2013 for treatment of Her2 positive breast cancer.19 The pharmacological properties of the resulting homogeneous TDCs were compared with Kadcyla and the TDCs demonstrated improved safety in both rat and cyno toxicity studies. Interestingly, the pharmacokinetic profile of the TDC was comparable to the Kadcyla benchmark. This led the researchers to conclude that the improved safety profile was likely due to removal of the higher DAR species present in Kadcyla rather than improved linker stability. A follow-up study by Pillow and coworkers utilized an oxime linker for conjugation of DM1 to trastuzumab through engineered cysteines on both heavy and light chains to generate homogeneous TDCs with four drugs/antibody.

46

Further improvements in efficacy and safety were observed for the

TDC versus Kadcyla. The improved properties were attributed to enhanced stability of the oxime linker, however, other factors such as different linkers (SMCC vs. MPEO or MPA), conjugated through different side chains (Lys vs. Cys), at different locations on the antibody, likely contributed to the observed differences in pharmacological properties. As a result, the relative contributions of homogeneity or linker stability to the observed improvements in therapeutic index could not be determined from these studies. In summary, the engineered cysteine approaches afforded homogeneous TDCs with superior therapeutic windows over conventional ADCs. The improvements were attributed primarily to improved linker stability and elimination of high DAR species that are present in heterogeneous ADCs. Engineered cysteine conjugated through non-maleimide linkers have also been reported. For example, Casi and coworkers introduced cysteine residues at the N-termini of antibody heavy and light ACS Paragon Plus Environment

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chains to enable site-specific conjugation with aldehydes. The engineered antibody was conjugated with a cemadotin aldehyde derivative. The conjugation process resulted in efficient formation of a cleavable thiazolidine linkage between the payload and the antibody intended to slowly release the payload in vitro (Scheme 2C). The ADCs contained four payloads per antibody and demonstrated moderate potency against antigen expressing cells in vitro, but their in vivo efficacy was not reported. 47 An important lesson learned from engineered cysteine approaches for synthesizing homogeneous ADCs was the discovery that the location of the conjugation site can have a dramatic impact on ADC activity. Shen and coworkers introduced cysteine mutations into three different sites on trastuzumab heavy and light chains.

48

The resulting thiomabs were used to construct homogeneous ADCs via

conjugation with an auristatin payload (MC-VC-MMAE) that contained a cleavable, self-emolative dipeptide maleimide linker analogous to that used in Adcetris.27 The three conjugation sites (LC-V205C, HC-A114C and Fc-S396C) were selected based on differences in solvent accessibility and local charge. All three thio-trastuzumab-MC-VC-MMAE ADCs demonstrated comparable homogeneity to each other with DARs ranging from 1.7 – 1.9 drugs/antibody and varied only in their conjugation sites. Remarkably, the ADCs demonstrated substantially different pharmacological properties in vivo, attributed primarily to differences in linker stability. Native LC/MS analysis of the ADCs revealed that linker stability correlated with the rate of maleimide hydrolysis to a ring-opened form that was less prone to premature release of the payload. The observed differences in the rates of hydrolysis were postulated to result from subtle variations in the microenvironments at different conjugation sites. This hypothesis was later confirmed by Tumey and coworkers who synthesized heterogeneous trastuzumab ADCs using maleimide linkers.

49

The ADCs

were subsequently hydrolyzed in vitro to the ring-opened isoform and their pharmacological properties were compared with analogous ADCs containing conventional (ring-closed) maleimide linkers. As expected, the ADCs containing the ring-opened form demonstrated improved stability and superior efficacy over the conventional (ring-closed) ADCs. Overall, the results demonstrated that the conjugation site can significantly effect ADC activity, suggesting that optimal conjugation sites might be different for each ADC. Several important breakthroughs in ADC technology have been made through use of engineered cysteines. For example, researchers at Seattle Genetics recently reported SGN-CD33A, an ADC that contains a highly potent pyrrolobenzodiazepine dimer (PBD) payload that is 10-100 times more potent than current tubulin inhibitor payloads such as MMAE or DM1.

50

Earlier attempts to construct

conventional ADCs using PBD payloads often resulted in aggregate formation due to poor water solubility of PBDs. Conjugation via engineered cysteines enables the synthesis of homogeneous ADCs ACS Paragon Plus Environment

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containing only two PBDs per antibody and reduces aggregation to an acceptable level. Moreover, the resulting ADC had superior potency over a well-known anti-CD33 ADC (Mylotarg) in AML tumor models with a multidrug-resistant phenotype. Positive results were also reported for an anti-CD70 ADC containing similar PBD payloads conjugated through engineered cysteines at position 239 on the heavy chains.

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The engineered cysteine approach propelled both of these homogeneous ADCs into early

clinical development and preliminary results have been very encouraging.

Engineered non-natural amino acids Non-natural amino acids (nnAAs) have been used as alternatives to cysteine for providing sitespecific conjugation sites.52-54 The nnAA approach offers several advantages over engineered cysteines ACS Paragon Plus Environment

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due to the diversity of different side chains that can be introduced as potential conjugation sites. For instance, liabilities associated with engineered cysteines and maleimide linkers can be eliminated and new payloads can be tested that might be incompatible with conventional methods for cysteine conjugation. In addition, the incorporation of nnAAs with different side chains on antibody heavy and light chains would enable payloads with different mechanisms of action to be conjugated to the same antibody. A number of different processes in which non-natural amino acids were used to afford ADCs with improved properties have recently been reported. For example, stop codon suppression technology (EuCODE) was used to express antibodies containing p-acetyl phenylalanine (pAF) at position 115 on the heavy chains. Site-specific conjugation of the pAF side chains to an auristatin payload (MMAD) containing an alkoxyamine linker forms a stable oxime linkage with the antibody. The conjugation process affords ADCs with 2 drugs/antibody (Scheme 3A).55 The resulting non-natural amino acid drug conjugate (NDC) was compared to an analogous thiomab drug conjugate (TDC) containing engineered cysteines instead of pAF at identical locations on the heavy chains. The TDC payload contained an analogous oxime linker to the NDC, but a maleimide group was added to enable conjugation with cysteine. Differences in activity between the NDC and TDC could therefore be attributed to the presence (or absence) of a thio-succinimide link to the antibody. The results demonstrated that the homogeneous NDC out-performed the TDC in vivo, attributed in part, to the absence of a thio-succinimide group in the NDC. Engineered non-natural amino acids provide new options for linker chemistry that are not possible with conventional conjugation methods or engineered cysteines. For example, Zimmerman and coworkers used non-natural amino acids in combination with a cell free expression system to incorporate p-azidomethyl L-phenylalanine (pAMF) into dozens of different sites on Trastuzumab.56, 57 The pAMF amino acid was designed for conjugation to alkyne linkers via strain-promoted azide-alkyne cycloaddition copper free click chemistry (Scheme 3B). The results were consistent with previous approaches in that ADC activity was highly dependent on the conjugation site. Antibody expression and conjugation efficiency were also affected by the location of the pAMF, suggesting that optimal conjugation sites may be different for each antibody. The data supported this suggestion, because the HC-Ala114 conjugation site previously used for making NDCs and TDCs was found to be inferior to Ser136 based on antibody expression and conjugation efficiencies. Further studies are needed to determine whether conjugation sites will remain optimal when applied to different antibodies.


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Alternative DNA targeting payloads such as pyrrolobenzodiazepine dimers (PBDs) have been successfully conjugated to antibodies through non-natural amino acids to yield homogeneous NDCs with two drugs/antibody.58 For example, VanBrunt and coworkers used a cell-based mammalian expression system to produce variants of a Her2 specific antibody (4D5) in high yield (1.7 g/liter). The variants contained a non-natural lysine analog (N6-2-azidoethoxy- carbonyl-L-lysine) modified with a terminal azide group. The azido-lysine derivative was engineered into positions on either chain of the antibody (HC-274 or LC-70) to enable site-specific conjugation via copper assisted alkyne cycloaddition (CuAAC) click chemistry (Scheme 3C). Auristatin F or PBD payloads containing alkyne linkers were conjugated with high efficiency (>95% conversion) after 4 hours at room temperature to afford NDCs with 1.9 drugs/antibody. The conjugation process forms a stable triazole linkage with the antibody. NDC stability was determined in vivo via single intravenous injections in rats and found to be comparable to ACS Paragon Plus Environment

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unconjugated trastuzumab. In addition, the PBD NDC demonstrated superior efficacy over an analogous auristatin NDC in Her2 positive BT474 tumor bearing mice after three weekly doses at 1 mg/Kg. This result further validated the use of PBDs as alternatives to conventional tubulin inhibitors.  Enzyme Mediated Approaches Transglutaminase Alternative methods for site-specific conjugation have been reported in which enzymes are used to site-specifically modify antibodies with unique functional groups for conjugation. For example, Strop and coworkers introduced a microbial transglutaminase (mTG) recognition sequence tag (LLQGA) into ninety different positions on an anti-EGFR antibody. 59,60 The glutamine tag served as an acyl donor for enzymatic ligation to primary amines catalyzed by mTG (Scheme 4A). The tagged antibodies were enzymatically conjugated with MMAD payloads containing cleavable (Ac-Lys-vc-MMAD) or noncleavable (amino-PEG6-MMAD) primary amine linkers. Twelve sites were considered adequate for ADC synthesis based on conjugation efficiencies, and two sites (one each on the heavy and light chains) were selected for further evaluation. The glutamine tags were engineered into different antibodies and consistently afforded ADCs with DARs > 1.8 drugs/antibody determined by native MS analysis.

61

ADCs were prepared from the cleavable Ac-Lys-VCP-MMAD payload using transglutaminase and their pharmacological properties were evaluated in vivo. In general, the mTG modified ADCs had comparable efficacy to control ADCs synthesized via conventional methods. In addition, they demonstrated improved stability and reduced toxicity in rodents, attributed in part to the formation of stable amide linkages with the antibody. Payloads containing non-cleavable linkers were also reported to conjugate with high efficiency, but their potency was not reported. The non-cleavable ADCs could have been informative controls since drug release would likely be restricted to antibody degradation. Instead, the control ADCs used in this study were heterogeneous, had higher DARs and contained payloads conjugated to different sites. As a result, the relative impact of the amide linkage resulting from mTG mediated conjugation remains uncertain. Alternatively, transglutaminase can be used without introducing a sequence tag.

62

Early studies

showed that deglycosylation of antibodies at position N297 enables site-specific bacterial transglutaminase (BTG) mediated conjugation to the native glutamine at position Q295. 63 Lhospice and co-workers later produced aglycosylated variants of cAC10 (the anti-CD30 antibody in Adcetris) with an N297Q mutation. 64 The mutation enabled site-specific conjugation of MMAE payloads to Q295 and


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Q297 on the heavy chains using BTG. The conjugation process afforded ADCs containing four payloads/antibody with 70% efficiency, but significant amounts of lower DARs were present. Nonetheless, the pharmacological properties of the transglutaminase modified ADCs were comparable to Adcetris. Overall, the study results expanded the transglutaminase approach to include ADCs with 4 drugs/antibody, and demonstrated that aglycosylated antibodies could be used without adverse effects. Formylglycine generating enzyme (FGE) Alternative enzyme mediated approaches have been used to construct homogeneous ADCs. Drake and coworkers introduced the recognition sequence (CXPXR) for formylglycine generating enzyme (FGE) into eight different sites on a generic IgG1 antibody (1 light and 7 heavy chain sites). 65 66 67 The approach is reminiscent of engineered cysteine approaches except that cysteine is introduced as a pentapeptide insertion rather than a single point mutation. Treatment of the mutant antibodies with FGE results in site-specific conversion of cysteine to formylglycine. The inserted peptide reduces the number of potential conjugation sites relative to other recombinant approaches due to structural constraints, but the aldehyde functionality enables new conjugation chemistries to be explored. Two of the heavy chain labeled sites resulted in highly aggregated antibodies and one was predicted to be immunogenic. Three of the five remaining sites were selected for evaluation using trastuzumab as a benchmark antibody. The aldehyde-tagged antibodies were site-specifically conjugated via hydrazino-iso-Pictet-Spengler (HIPS) chemistry to a maytansine payload containing an appropriately modified hydrazine linker (Scheme 4B). The pharmacological properties of the resulting homogeneous ADCs were compared to Kadcyla, a heterogeneous trastuzumab ADC with an analogous maytansine payload. Consistent with previous approaches, the homogeneous trastuzumab ADCs demonstrated comparable potency, improved stability and reduced toxicity in vivo compared to Kadcyla. Contrary to results from previous approaches, the conjugation site did not significantly impact ADC efficacy and minimal differences in tumor growth inhibition were observed in xenograft tumor models. The differences between Kadcyla and the FGE tagged ADCs were attributed to the presence of high DAR species present in Kadcyla. Although Kadcyla contains an analogous maytansine payload, it is a heterogeneous ADC and is prepared using different linker chemistry conjugated to lysines at different sites; properties which likely contributed to the observed differences in activity of the ADCs. To simplify the overall aldehyde tagging approach, antibody expression was carried out in cells overexpressing FGE. Efficiencies for the cysteine to formylglycine conversion ranged from 86% to 98% depending on the conjugation site. Conjugation efficiencies between the tagged antibody and the hydrazine payload were typically 75% or higher, although 8-10 equivalents of the payload were required to obtain ADCs with two drugs/antibody. Moreover, preparative hydrophobic interaction ACS Paragon Plus Environment

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chromatography (HIC) was required to remove unconjugated antibody in the final purification step, which raises questions regarding scalability of the process to a level required for clinical development. Sortase A Alternative enzyme mediated approaches have been used to generate homogeneous ADCs. For example, Beerli and coworkers engineered a recognition sequence for a transpeptidase (sortase A) to the C-termini of the heavy and/or light chains of antibodies.

68

Sortase A catalyzes transfer of polyglycine

substrates to the C-terminus of the pentapeptide sequence motif, LPXTG, resulting in a stable amide linkage with the antibody (Scheme 4C). ADCs were constructed via conjugation of engineered trastuzumab and cAC10 variants containing an LPETG recognition motif with MMAE or DM1 payloads containing pentaglycine linkers. The resulting ADCs had improved homogeneity vs. conventional Kadcyla and Adcetris controls and contained approximately 1.8 drugs/antibody after purification by affinity chromatography. Notably, the homogeneous trastuzumab ADC demonstrated comparable efficacy to Kadcyla in Her2 expressing xenograft tumors despite have a lower DAR. Additional studies with ADCs containing auristatin payloads are ongoing to demonstrate the versatility of the methods for use with alternative payloads.

Glycosyltransferases Additional enzyme-mediated approaches to site-specific conjugation have been reported which do not require recombinant engineering. For example, Zhou and coworkers used glycosyltrasferases to incorporate terminal sialic acid moieties into glycans linked to the native Asn297 glycosylation site in trastuzumab and two other antibodies.

69

Mild oxidation of the engineered sialic acid groups with

sodium periodate yields two aldehyde groups for site-specific conjugation to appropriately modified payloads. The glyco-engineered antibodies were conjugated to auristatin payloads (MMAE and MMAD) containing amino-oxi linkers resulting in formation of a stable oxime linkage with the antibody (Scheme 4D). Unlike other approaches discussed so far, the method does not require recombinant antibody engineering, but relatively large quantities of the linker-payload (24 equivalents) were required to produce ADCs containing 1 or 2 drugs/antibody. The in vivo stability and pharmacokinetic properties of the ADCs were not reported, but they demonstrated comparable activity to conventional controls in xenograft tumor models.


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Endoglycosidase Another non-recombinant approach to site-specific conjugation that involves glycan remodeling at Asn297 was reported recently by van Geel and coworkers.70 The multi-stage process begins by treatment of the antibody with endoglycosidase to remove core GlcNAc moieties. This enables sitespecific attachment of azide-modified GalNAc analogs using glycosyltransferases. The resulting azidelabeled antibody is then conjugated to payloads with bicyclononyne (BCN) linkers via copper-free click chemistry (Scheme 4E). A variety of different linker-payload constructs were synthesized using five different payloads combined with cleavable or non-cleavable linkers. Conjugation efficiencies were typically >95% and the ADC (trastuzumab-BCN-PEG-DM1) outperformed Kadcyla in efficacy studies.  Linker-Based Approaches The engineered amino acid and enzyme mediated approaches for site-specific conjugation have been successful in producing ADCs with improved homogeneity and led to significant improvements in other ADC properties such as stability, potency and safety. Moreover, most of the methods discussed thus far yielded ADCs with comparable or superior therapeutic windows when compared to conventional heterogeneous benchmarks. The relative impact of homogeneity on ADC activity remains uncertain however, because the homogeneous ADCs often contained different payloads and conjugation sites than those used in the benchmark ADCs. Conjugation methods that leverage the same conjugation sites as conventional ADCs should enable the impact of homogeneity on ADC activity to be determined with greater confidence because other variables can be eliminated. The majority of the linker-based processes for constructing homogeneous ADCs utilize interchain cysteines for conjugation to afford homogeneous ADCs with four or eight drugs/antibody. The processes are chemically driven and differ from previously discussed processes in that they are focused on linker modifications. As a result, they can be applied to existing „off the shelf‟ antibodies and don‟t require recombinant antibody re-engineering or unconventional expression systems.

Hydrophylic linkers As discussed previously, ADCs with eight payloads/antibody conjugated through interchain cysteines were shown to possess sub-optimal pharmacological properties, attributed in part to the hydrophobicity of the conventional payloads that were used for their preparation. With this in mind, researchers at Seattle Genetics hypothesized that hydrophylic linkers and payloads would enable construction of fully loaded ADCs (DARs = 8 drugs/antibody) without compromising other desired properties. Earlier work by Doronina et al. had shown that dipeptide linkers could be used for conjugating auristatins through ACS Paragon Plus Environment

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their C-terminus to yield heterogeneous ADCs with improved potency over conventional auristatin ADCs.71 Lyon and coworkers synthesized similar auristatin derivatives with reduced hydrophobicity by replacing the C-terminal phenylalanine in auristatin F with a more hydrophylic amino acid, threonine. The resulting derivative (auristatin T) was then linked through the C-terminus to a hydrophylic dipeptide maleimide linker (Scheme 5A).

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The hydrophylic payload was then conjugated to an anti-CD70

antibody (h1F6) to afford homogeneous ADCs containing eight drugs/antibody. The fully loaded ADCs were compared to ADCs containing conventional linkers (MC or MC-VC-PAB) and payloads (MMAF) and demonstrated slower clearance and improved efficacy over the conventional ADCs. The results led the authors to conclude that; “reducing hydrophobicity of homogeneous ADCs improves pharmacokinetics and therapeutic index”, as reflected in the title of the article. ADC hydrophylicity correlated with improved pharmacological properties, however, the ADCs compared in the study, and they all contained different payloads, connected to different linkers in different orientations (N or C terminus). Since these factors would also have a significant effect on the overall properties of the ADCs, the relative contribution of reduced hydrophobicity to the improved therapeutic index could not be determined with confidence. 73

Bis-alkylating linkers Most linker based approaches for synthesizing homogeneous ADCs utilize bifunctional linkers designed to cross-link antibody interchain cysteines and afford homogeneous ADCs containing four drugs/antibody. One example of this approach was reported by Badescu and coworkers who synthesized bis-sulfone linkers designed to cross-link two cysteines and form a 3-carbon bridge (Scheme 5B).74, 75 The bis-sulfone cross-linking group was attached to MMAE through a cleavable PEG spacer and conjugated to trastuzumab to afford ADCs that were 78% DAR4. The resulting ADCs were more stable than conventional maleimide ADCs under various conditions and were moderately potent against Her2 positive cells in vitro. Efficacy studies were performed, and the bis-alkylated ADCs demonstrated superior potency to unconjugated trastuzumab, although multiple high doses (>10 mg/Kg) were required for tumor growth inhibition. In a follow-up study, Godwin and coworkers used a similar payload to construct ADCs containing predominately 1, 2, 3 or 4 drugs per antibody by changing the stoichiometry of the conjugation reaction.76 The ADCs were purified by preparative hydrophobic interaction chromatography (HIC) to remove undesired DAR fractions and tested in a BT474 xenograft tumor model. ADC efficacy correlated with increased DAR and again, multiple high doses (> 10 mg/Kg) were required for tumor ACS Paragon Plus Environment

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growth inhibition. A second study in JIMT-1 xenografts was performed and the bis-alkylated ADCs demonstrated improved potency compared to T-DM1. Since neither unconjugated trastuzumab nor analogous heterogeneous ADC controls were included in these studies, the relative impact of the bisalkylating linkers on the overall therapeutic index of the ADCs could not be determined.

Next generation maleimides (NGMs) Alternative reagents have been used for cross-linking interchain cysteines with improved efficiency over the previously discussed bis-sulfone linkers. Researchers at UCL (University College of London) published a series of papers in which substituted maleimides were shown to be highly efficient cysteine cross-linking reagents.77-79 Early applications for substituted maleimides included disulfide protection, protein pegylation, and fluorescent labeling.80-82 Moody and coworkers later reported that bromomaleimide-linked bioconjugates are cleavable in mammalian cells,

83

based applications with substituted maleimides have recently been reported.

84-87

and numerous antibody-

For example, Caddick and coworkers at University College of London (UCL) reported the synthesis of homogeneous trastuzumab ADCs using a three-step process. Interchain disulfides were reduced with TCEP followed by addition of an N-propargyl-3,4-dithiophenylmaleimide linker to form a dithiomaleimide linkage with the antibody. Addition of an azido-doxirubicin derivative resulted in formation of a triazole linkage with the payload (Scheme 5C). The process was applied to trastuzumab and afforded homogeneous ADCs with four drugs/antibody. The ADCs demonstrated comparable antigen binding affinity compared to the parent antibody, but in vivo pharmacological properties were not reported. Unlike conventional methods for cysteine conjugation, covalent bonds between antibody H and L chains are maintained which is expected to improve the stability of the cross-linked ADC.88

Dibromopyridazinediones Maruaini

and

coworkers

later

published

a

similar

cross-linking

approach

using

a

dibromopyridazinedione linker that contained dual orthogonal alkyne functional groups. The linker was designed to enable chemo-selective conjugation with two different azide derivatives using click chemistry (Scheme 5D). Doxirubicin and a fluorophore, both modified with azide groups, were conjugated sequentially to Herceptin resulting in an ADC with 4 payloads and 4 fluorophores. The dual labeled ADC was stable in plasma and selectively killed Her2 positive BT474 cells at µM concentrations. Although the pharmacological properties of the ADCs in vivo were not reported, the results suggested that a similar strategy could be used for conjugating two different payloads to an antibody with a single linker. 89 ACS Paragon Plus Environment

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Dibromomaleimides In order to determine the effect of interchain cysteine cross-linking on the in vivo properties of ADCs, Behrens and coworkers synthesized a derivative of monomethyl auristatin F that contained a dibromomaleimide (DBM) linker instead of a conventional maleimide.

90

The DBM-MMAF payload

was designed to crosslink interchain cysteines to form a dithiomaleimide linkage with the antibody (Scheme 6). The DBM-MMAF payload was conjugated to trastuzumab and a novel anti-CD98 antibody to afford homogeneous ADCs with four drugs/antibody.

91

The ADCs selectively bound to antigen


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expressing cells in vitro with comparable affinity to the parent antibodies and inhibited growth at sub nM concentrations. The pharmacological properties of the cross-linked ADCs in vivo were compared to analogous heterogeneous ADCs synthesized using conventional maleimide (MC) linkers. The results demonstrated that the DBM linkers yield homogeneous ADCs directly from a variety of different antibodies without recombinant engineering. Importantly, the DBM cross-linked ADCs demonstrated improved pharmacokinetics, safety and efficacy over analogous conventional heterogeneous ADCs.

The protocol for DBM conjugation requires fewer steps than most previously discussed methods, and consistently affords ADCs with > 90% DAR 4. The reduction and conjugation processes can be performed sequentially in one pot at room temperature in less than 3 hours. Unlike other methods for generating homogeneous ADCs, excess reagents are unnecessary and the process is easily scalable to gram quantities. Buffer exchange or membrane filtration of the crude conjugation mixture affords highly pure ADCs. The DBM cross-linking approach was the first study in which homogeneous ADCs were directly compared to analogous heterogeneous ADCs containing identical linkers and payloads conjugated to identical sites. Since other variables known to affect ADC properties were effectively removed from the study, the relative contributions of homogeneity and interchain cysteine cross-linking could be accurately determined. The results demonstrated that interchain cross-linking with DBM does not adversely affect ADC activity in vivo relative to conventional methods. In addition, the results provided convincing evidence that homogeneous ADCs are superior to their heterogeneous counterparts and validated previous efforts to construct homogeneous ADCs with defined DARs.


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 Discussion All of the processes reviewed here were successfully used to construct ADCs with improved homogeneity over ADCs synthesized using conventional methods. A majority of approaches utilize recombinant antibody engineering to introduce unique functional groups for site-specific conjugation. The unique functional groups were introduced either as point mutations for cysteine and non-natural amino acids, or as enzyme recognition tags. These recombinant engineering approaches offer several potential advantages over non-recombinant approaches. For example, engineered cysteines can be incorporated into dozens of different sites with minimal impact on the functional properties of the antibody. This enables ADCs to be optimized for conjugation efficiency, linker stability, and potency. Engineered non-natural amino acids offer additional advantages due to the diverse array of different functional groups that can be introduced. Furthermore, non-natural amino acids enable a variety of new linker chemistries to be investigated that are not possible with conventional conjugation processes. The flexibility offered by recombinant processes may also represent their greatest challenge. The importance of the conjugation site for ADC activity is well established, but additional factors should be considered before selecting a development candidate. Potential effects on antibody expression, conjugation efficiency, linker stability, aggregation, and other factors need to be considered before selecting a specific conjugation site. These factors can ultimately determine the success or failure of an ADC development program. Since antibodies share many of the same properties, it seems likely that optimal conjugation sites will be identified that are broadly effective when used with different antibodies. Other potential challenges for processes involving antibody engineering include increased development time and costs, immunogenicity of engineered sequence tags, scalability and use of novel linkers and payloads that are not yet clinically validated. In addition to homogeneity, improvements in other ADC properties such as potency, stability and half-life were observed. In fact, many of the homogeneous ADCs derived from these processes outperformed conventional heterogeneous ADCs in efficacy and safety studies. Much of their success has been attributed to elimination of high DAR species present in conventional ADCs. In general, experimental results are consistent with this conclusion and many would agree that substantial progress has resulted from these efforts to improve ADC homogeneity. Ironically, the relative contribution of homogeneity to the improved properties of the engineered ADCs could not be determined from most studies because other factors known to effect ADC activity could not be ruled out. For instance, recombinant approaches for making homogeneous ADCs were designed to introduce conjugation sites in different locations from those used in conventional methods. Since it is now wellACS Paragon Plus Environment

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established that “location matters”, the observed differences in activity between TDCs (or NDCs) and the conventional ADC controls could result from different conjugation sites, rather than from elimination of high DAR species. Enzyme mediated approaches face similar challenges when comparing homogeneous and heterogeneous ADCs because the conjugation sites are different. Other variables such as linker type (cleavable or non-cleavable) and payload (maytansine or PBD) need to be carefully controlled before reaching conclusions about the benefits of homogeneity. Linker based processes are more suitable for comparing homogeneous ADCs with conventional heterogeneous ADCs because they utilize the same conjugation sites. Once other variables that might impact ADC activity were carefully controlled, the relative benefits of homogeneity were revealed for the first time and the results confirmed that efforts to improve ADC homogeneity have been a worthwhile endeavor. Most of the processes reviewed here are still in early phases of clinical development. All of the methods have advantages and limitations that will ultimately decide which approach will become the preferred process for manufacturing homogeneous ADCs. It is not yet clear which process will rise above the others as a preferred method, but all of these approaches have contributed valuable information to our knowledge base and resulted in ADCs with improved pharmacological properties over conventional heterogeneous ADCs. Our future challenge will be to apply this knowledge to develop ADCs that will be more effective as therapeutic agents. Our ability to synthesize homogeneous ADCs provides another reason to be optimistic about the future of ADCs.  References 1. Chari, R. V. J.; Miller, M. L.; Widdison, W. C., Antibody-Drug Conjugates: An Emerging Concept in Cancer Therapy. Angew. Chem., Int. Ed. 2014, 53, 3796-3827. 2. Beck, A.; Reichert, J. M., Antibody-drug conjugates in cancer therapy. Annu. Rev. Med. 2014, 64, 1529. 3. Sievers, E. L.; Senter, P. D., Antibody-drug conjugates in cancer therapy. Annu. Rev. Med. 2013, 64, 15-29. 4. Goldmacher, V. S.; Chittenden, T.; Chari, R. V. J.; Kovtun, Y. V.; Lambert, J. M., Antibody-drug conjugates for targeted cancer therapy. Annu. Rep. Med. Chem. 2012, 47, 349-366. 5. Gerber, H.-P.; Koehn, F. E.; Abraham, R. T., The antibody-drug conjugate: an enabling modality for natural product-based cancer therapeutics. Nat. Prod. Rep. 2013, 30, 625-639. 6. Flygare, J. A.; Pillow, T. H.; Aristoff, P., Antibody-drug conjugates for the treatment of cancer. Chem. Biol. Drug Des. 2013, 81, 113-121. ACS Paragon Plus Environment

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Processes for Constructing Homogeneous Antibody Drug Conjugates

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