See also detection of activatable antibodies under Diagnostics. See also Conjugation and Linkers See also Particular Contaminants under Antibody Purification

Cleavable Linkers:

Problem of stability with cleavable linekrs:

A peptidic linker moeity that is a specific substrate for acid hydrolases can be leaved preferentlly inside a lysosome. Or,  a redox potential sensitive linker moeity may be preferentlly cleaved in response to a difference in redox potential. Nevertheless a certain amount of premature clavage of the linker moeity is virtually unavoidable. (Santi, US 7,541,330). 

The rapidly expanding field of antibody-drug conjugates for cancer therpay has relied on the proeprties of linkage chemistries which ideally would offer both a stable toxin attachment to antibdoy while in the systemic ciruclation and an efficent release by intracellular protease. 

Types of Cleavable Linkers:

Cleavable linkers fall into four categores: (1) acid labile (e.g., hydrazone) linkers, which are subject ot low pH acid clevage in lysosomes (2) disulfide linkages which dissociate in the low redox potential of early endosomes, (3) beta-glucuronide linkers which are cleaved by lysosomal beta-glucuronidase and (4) peptide linkers which are cleaved proteolytically by lysosomal proteases. (Chamow “Manufacturing challenges of therapeutic antibody -drug conugates, BioProcess Internaitonal, September 2023).

The valine-citrulline dipeptide linker introduced by Seagen is the indsutry’s most frequently used peptide linker. Dislfide and hydrozone linkers are useful because they enable intracellular durg release while minimizing off target toxicity. Dislfide and hydrazone linkers offer stability and selective intracellular clevage, with the later enalbing tumor specific release through pH sensitivity. (Chamow “Manufacturing challenges of therapeutic antibody -drug conugates, BioProcess Internaitonal, September 2023).

Heidtmann discloses amino acid sequences which can be cleaved by proteases which are formed in tumors or by tumor cells or inflammatory cells. The nucleic acid sequence of this sequence (designated as “C”) is inserted, for example, into the nucleic acid sequence of the naturally occurring precursor protein BSD, where S is the naturally occurring cleavage sequence of the relevant active compound protein B) in place of the cleavage sequence S such that this recombinant nucleic acid expresses protein BCD. The peptide D binds to the activate compound (B) by way of C and inactivates the active compound B by means of this binding.

Morgan (10/499611) discloses cleavage sites for an enzyme present at sites of inflammation which link at one end a complement regulatory protein (CReg) and at the other end a carrier protein which is at least an antibody Fc region which serves to render the CReg inactive until the CReg is adjacent to a target site where cleavage occurs.

Rodwell (US 4671958) discloses linkers which are susceptible to cleavage by serum complement enzymes. The linker groups are covalently attached to specific sites on an antibody which are directed against any desired target antigen such as a tumor.

–Acid-sensitive linkers:

Acid sensitive hydroazone groups in acid labile linkers remains stable in systemic circulation (pH 7.5) and gets hydrolyzed in lysosomal (pH 4.8) and endosomal (pH 5-6) acidic tumor micro-environment upon internalization in the targeted cells. 

–Glutahtione-sensitive disufide linkers:

Glutathione is a low MW thiol which is present in the cytoplasms and extracellular environment. In tumor cells elevated levels of thiols are found during stress conditions such as hypoxia. The differenc ein gluthatione concetnraiton in cytoplams and extracellular environment can be implemented as a selective delivery of the drug payload to target tumor via breakdown of disulfide linkers. Besides glutathione, intercellular protein disulfide isomerase is capable of reducing disulfide bonds. Two cysteine residues int he active site of this enzyme governs the thiol-disulfide exchange reactions within substrates. Maytansinoid drug conjugates ahve been widely meployed for disulfide bonds . 

Lysosomal protease-sensitve peptide linekrs:

Tumor cells have higher expression of lysosomal proteases like cathepsin B than normal cells. Cathepin B sensitve peptide linekr conjugated ADCs selectively bind to and gets internalized into tumor cells via receptor mediated endocytosis. Proteases are inactivated in serum in presence of a high pH and different serum protease inhibitors. This makes the peptide linker stable in systemic circulation and only to be cleaved upon internalization in tumors. The FDA approved Adecetris, cathpesin B senstive valine-citruline linker is found to be superior to hydrozone linker. 

–Cleavable diphosphate linkers

Brixner (US 5094848) discloses a method of targeting a pharmaceutical agent to a desired site by administrering the agent bound by a cleavage diphosphate linked to a targeting moiety. The targeting moiety can include antibodies.

–Protease-Cleavage linkers:

—-cleavage by cathepsins: 

—–Valine-citrullin (VCit) dipeptide linkers connecting a payload with a p-aminobenzyloxycarbonyl group are standardcleavable linkers widely used in many successful ADC. VCit linkers are cleaved by cathepsins upon internalization of ADCs by target cancer cells, resulting in traceless release of payloads. VCit linkers are stable in cynomolgus money and human plasma. However, these linkers can be hydrolyzed in mouse plasma. The linker instaility in mouse plasma can be ameliorated by slecting the linekr attachment sties within an antibody and limiting the lengh of the VCit linker to minimize the exposure of the vulnerable moiety to extracellular enzymes. A glutamic acid valine-citrulline tripeptide sequences has also been shown to proide exceptionally high long term stability in mouse and human plasma while retaining the capacity to release the payload upon cathepsin mediated clevage. (Anami, “Glutamic acid-valine-citrulline linkers ensure stability and efficacy of antibody-drug conjugates in mice” Natural Communications, (Dec 2018, 9(1))

–matrix metalloprotease (MMP)

Shyra (US 20190185561) discloses a emthod for treating a CD47 expressing cancer which includes admisntiering a masked antibody wherein each masking domain of the masked antibody includes a protease-cleavaeg linekr and wherein the protease cleavage linker includes matrix metalloprotease (MMP) clevage site. 

Types of non-cleavagle Linkers:

A linker can also be a non-cleavable linker such as maleimido-alkylene or maleimide-aryl linker that is directely attached to a therpaeutic agent and relased by proteolytic degradation of the antibody. Shyra (US 20190185561)

Cleavagle Linkers as part of Activatable antibodies (Probodies):

Probodies are antibodies that are “masked” to prevent binding to normal tissue and are “un-masked” to bind tumors by disease specific protease activity. This technology is applicable to all antibody formats includeing naked antibodies, antibody drug conjugates, bispecific antibodies (T cell engagers) and engineered T cells (CART cells). (Bryan Irving, “Probodies empower a new generation of antibody immunotherapies” Cytomx Therapeutics). 

Probody therapeutis are a novel class of recombinant antibody based therapeutics that target antibody activity to the tumor by tacking advantage of the dysregulation of protease in diseased tissues. The eky components are two peptide sequenced encoded on the N temrinus of the light chain of antibodies collectively called the Prodomian. The first sequence is a “masking” peptide which physically blocks the ability of the antibody to bind antigen. This sequence is connected to teh rest of the L chain by a second peptide sequence designed to be preferentially cleaved by proteases with increased acitvity in tumors. Probody drug conjugate components include a parental antibody; the Prodomain which includes a masking peptide linked to the N terminus of the L chain via a protease substrate and fianlly the linker/toxin. In theri inactive form, PDCs have reduced binding for their antigen and upon activation by proteases, recover the binding equivalent to that of the parental antibody. (Sagert “Targeting Drug conjugates to the tumor microenviornment: probody drug conjugates” Cancer Drug Discvoery and Development” 2018).

Masking Moiety with Cleavable Moiety (activatable Binding Polypeptides): 

Daugherty (WO 2009/025846) discloses activatable binding polypeptides (ABPs) which contain a target binding moeity (TBM), a masking moiety (MM) and a clevable moiety (CM) where the CM is positioned in the ABP such that in a cleaved state in the presence of a target, the TMB binds the target and in an uncleaved state in the presence of the target, binding of the TBM to the target is inhibited by the MS. These ABPs exhibit an “activable” conformation such that at least one of the TMBs is less accessible to target when uncleaved than after cleavage of the CM in the presence of a cleaving agent capable of cleaving the CM. In further emobdiments, the MM inhibits binding of the TMB to the target via steric hindrance when the ABP is in an uncleaved state. In other embodiemnts, the MM comprises a cysteine residue and steric hindrance is acheived via a disulfide bond linkage between said cysteine residue an an additional cysteine residued adjacent to or with the TBM. The MM is an amino acid sequence that, when the CM o e the ABP is intact (i.e., unlceaved by a corresponding enzyme and/or containing an unreduced cystein-cysteine disulfide bond), the MM interfers with binding of the TBM to its target. In certain embodiments, the CM includes a protease substrate such as a caspase or a matrix metalloprotease (MMP) substrate (e.g., a substrate of MMP-1). Exemplary ABPs include those having a TMB that binds VEGF to inhibit VEGF activity at the tumor site. 

Menendez (WO 2013163631) discloses activatable antibodies which include an antibody that specifically binds EGFR coupled to a masking moiety (MM) such that ocupling of the MM reduces the ability of the antibody to bind EGFR. The MM is coupled via a sequence that includes a substrate for a protease such as a protease that is co-localized with EGFR at a treatment site in a subject. 

William (WO 2016/118629) disclsoes polypetpides taht include at elast a first clevable moeity (CM1) that is a substrate for at elast one matrix metalloprotease (MMP) and at least a seocnd cleavable moeity (CM2) that is a substrate for at elast one serine protease (SP) to activvatable antibodies that include these polypeptides. The CM1-CM2 substrates are useful in activatable antiboides taht include antibodies or AB fragmetns which include at elast one masking moeity (MM) linked to at least one antigen/epitope binding domain of the Ab such that coupling of the MM reduces the ability of the AB to bind its target. 

–anti-Epidermal Growth Factor Receptor:

Moore (US Patent No: 2015/0087810) discloses an activatable anti-EGFR antibody which incluees at least an AB that includes and H and L chain, a MM and a CM. 

Lowman (US 20150118254) discloses activatable antibodies that includ a masking moiety (MM), a cleavable moiety (CM) and an antibdoy (AB) that specifically binds to EGFR and to methods of making and using these activatable antibodies. 

Vasiljeva (WO 2014/107599) discloses activatable antibodies which bind EGFR and interleukin 6 receptor  which inclue a H and L chain, a CM and a MM. EGFR is highly expressed in H292 human non-small cell lunch cancer cells and efficacy of Cetuximab has been determined by multiple studies in xenograft tumor models. The H292 tumor tissue samples were slected to validate the activatable antibody in situ imaging. H292 xenograft tumor tissue sections were placed on a glass slide, rinsed two times followed by 30 min pretreatment of tissue with protease inhibitors or buffer only. Alexa Fluor-680 labelled activatable anti-EGFR anitbody and Alex Fluor 680 labeled non cleavalbe masked antibodyas positive and negative controls respectively were applied on the tissue and incubated for one hour. Fluorescnece microscopey analysis revleaed a positive staining of activatable anti-EGFR antibody that was abolished by the pretreatment of tissue section with protease inhibitors, indicating that the binding of the activatable antibody to the tissue sample is a result of the proteolytic event. 

–anti-Jagged antibodies:

Moore (US Patent No: 2015/0087810) discloses an activatable anti-Jagged antibody which incluees at least an AB that includes and H and L chain, a MM and a CM.

–anti-CTA-4 Activatable antibodies:

discloses improved anti-CTLA-4 antibodies which are activatable antibodies that specifically bind human CTLA-4 when activated. The activatable antibodies are used in methods of treating symptoms of a cancer. The antibodies can include a CM which is specific for a protease and can include a masking moiety (MM) which is an amino acid sequence that is attached to the anti-CTLA04 antibody and positioned such that the MM reduces the ability of the antibody to specifically bind CTLA-4. In order to identify masking moeities (MM) that reduce the binding of anti-CTLA-4 antibodies to their target, anti-CTLA04 antibody ipiliumumab was used to screen peptide libraries which consisted of two rounds of magnetic activated cell sorting (MACS) purificaiton followed by three rounds of fluorescence activated cell sorting (FACS). 

–anti-CD71 (transferrin receptor):

CD71 (tranferrin receptor) is an ideal probody drug conjugate (PDC) target not only becasue it efficiently internalizes adn devlier a cytotoxic payload intracellularly, but aslo becasue it is expressed at high levels both in many different tumor types as well as in dividing normal cells. Two key components of a probody therapeutic prodomain that reduce its binding to normal tissue and allow for its tumor-specific activaiton are 1) a mask that reeuces the ability of the antibody binding site to interact with target antigen and (2) a protease-activatable substrate that is cleaved in the TME resulting in removal of the mask. Moedultating mask strenth and substrate clevability can impoact efficacy and saftey of a CD71 targeting PDC in preclinical models. (Sing “Development a CD71 targeting probody drug conjugate (PDC) for activity in multiple solid tumor and lymphoma models and for tolerability in non-huan primates” Abstract, 2018

–anti-PD-1 activatable antibodies:

Chang (WO2017011580) discloses activatable antibodeis that bind to PD-1 coupled to a masking moeity (MM) such that coupling of the MM reduces the ability of the atibodny to bind PD-1. The MM is coupled via a cleavable moiety that includes sequence that fucntions as a substrate for a protease.

–-Particular Structures of Activatable Antibodies:

—-scFV-MM:

Fox (WO 2019213444) discloses an activatable antibody (AA) where the AA specifically binds to a target when activate and the AA includes the structure of at elast one scFv that includes a VL linked to a H wehre the VL is linked to the VH by a linker L3 and a prodomain which includes a MM coupled to the scFv which reduces binding of the scFv to its target wehn the AA is in an uncleaved state and a CM coupled to the scFv wehre the CM is a polypeptide that functions as a substrate for a protease. 

—-Multispecific Activatable Antibodies:

Irving (WO2015/013671) discloses multspecific antibodies which inlcude at least one masking moeity (MM) linked to at elast one antigen binding domain of the multspecific antibody. The MM can be coupled to the binding domain via a cleavalbe moeity (CM) which functions as a substrate for a protease. The antibodies are designed to engage immune effector cells. In some embodiments, the T cell engaging multispecific antibodies include a targeting antibody and a T cell engaging antibody such as anti-CD3 epsilon. In some embodiments of a T cell engaging multispecific activatable antibody one antigen is typically an antigen present on the surface of a tumor cells such as eGFR, Jagged, PD-L1, CD71 and another antigen is typically a stimulatory or inhibitory receptor present on the surface of a T cells, NK cells such as B7-H4, CD3, CD4, CD8, CD25, CD28. 

Moore (US Patent No: 2015/0087810) discloses multispecific activatable antibodies which recognize two or mroe different antigens which inlclude at least one masking moeity (MM) linked to at elast one antigen binding site of the antibodysuch that compling of the MM reduces the ability of the anitgen binding domain to bind its target. The MM can be coupoled to the binding domain via a cleavable moeity (CM) that functions as a substrate for a MMP protease. 

——T cell engagers:

Moore (US Patent No: 2015/0087810) discloses multispecific activatable antibodies that engage immune effector cells, also referred to as immune effector cell engaging multispecific activatable antibodies. In some embodimetns, the antibodies are designed to engage leukocytes. The T cell engaging antibody includes a first antibody that binds a first T cell engaging target which is attached to a masking moeity such that coupling of the MM redues the ability of the AB1 to bind the first target. The antibody can include a second antibody that binds a second, cancer related target wehre the AB2 is also attached to a MM such that coupling of the MM2 reduces the ability of the AB2 to bind the second cancer related target. The antibodiesonce activated by tumor assocaited proteases can crosslink and activate the T cell or NK cells expressed co-stimulatory receptors in a tumor dependent manner to enhance the activity of T cells that are responding to any tumor antigen via their endogenous T cell antigen or NK activating receptors. In some embodiments, the T cell engaging multspecific activatable antibody inlues an anti-CD3 epsilon scFv and a targeting antibody where at least one of the anti-CD3 epsilon scFv and/or targeting antibody is masked. 

With Drug (Antibody Drug Conjugates (ADC)):

discloses a method of treating cancer by adminstiering a combination of a masked CD47 antibdoy with an antibody drugate (ADS) where the antibody of the ADC specifically binds to a protein that is expressed on the extracellular surface of a cancer cell and the antibody is conjugated to a drug-linker that includes a cytotoxic agent. Shyra (US 20190185561)

Types of Cleavable Moeity/linkers used with Activatable Antibodies:

–Substrates for proteases:

Protease-cleavable linker:

Cunnigham (US 2022/0306727) disclsoes activatable antibodies which include a masking domain that incldues a protease-cleavable linker which is linked to the ehavy or light chain via the protease-cleavable linker. In some embodimetns, the protease cleavable linker includes a matrix metalloprotease (MMP), urakinase plasminogen, matriptaase or legumain cleavage site and a metalloprotease (ADMA clevage site or a caspase cleavage site. 

(Patrick, (US Patent Applicaiton No: 17/227,029, published as US 2021/0317188) disclsoes cleavable moiety for activatable antibodies which include a peptide that includes a substrate for at elast one protease. 

pH sensitive cleavable linker:

Cunnigham (US 2022/0306727)discloses that a cleavable linker can be pH senstive such as sensitive to hydrolysis at certain pH values. Typically, the pH senstive linker is hydrozable under acidic conditons. For example, an acid labile linker that is hydroable in the lysosome (e.g., a hydrazone, semicarbozone, thiosemicarbazone, cis-aconitic amid, orthoester, acetal, ketal or like) can be used. Such linkers are relatively stable under neutral pH conditions such as those in the blood, but are unstalbe at below pH 5.5 or 5, the approximate pH of the lysosome.

Disulfide linkers:

Cunnigham (US 2022/0306727) discloses other linkers whcih are cleavable under reducing conditions (e.g., a disulfide linekr) Disulfide linkers include those that can be formed using SATA, SPDP, SPDB and SMPT. 

Types of Linkers (e.g., for attachment of cytotoxic drugs) used with Activatable Antibodies:

Gly-Ser Linkers:

(Patrick, (US Patent Applicaiton No: 17/227,029, published as US 2021/0317188) discloses linkers suitable for sue in activatable antibodies that can be of any lenght. Typcially, the linker is a flexible linker that includes one or mroe amino acid residues selected from Gly, Ser, Ala and Thr, particularly Gly and Ser. Exemplary flexible linkers include glycine homopolymer.