Targeted therapy using monoclonal antibodies (mAbs) has reolutionized cancer treamtent, with several mAbs recognizing antigens expressed on the surfaces of tumor cells already haivng demonstrated their clinical potential. As antibodies against tumor-specific antigens often lack therapeutic activity, they alternatively can be covalently linked to cytotoxic drugs. In principle, selective delivery of cytotoxic agents should reduce the systemic toxicity associated with traditional small-molecule chemotherapeutics. (Junutul “Site-specific conjugaiton of a cytotoxic drug to an antibody improved the therpaeutic index” Nature biotechnology, 24(8), 2008).

Reports of targeting drugs using antibodies have appeared in the literature since 1958 with the simplest say of producing an immunoconjugate being to couple the drug directly to the antibody as by direct linkage between the functional group of the drug and one of the functional groups on the antibody, or alternatively by the interposition of a linker or spacer group between these two parts of the conjugate (Garnett, Advanced Drug Delivery Reviews 53 (2001) 171-216).

The key driver for the development of antibody-drug conjugates (ADCs) was to make use of the binding specificity of the antibodies for cell surface associated antigens to bring the cytotoxic agents (small molecular drugs or toxins) to the desired cell population. Specific binding of ADCs to cell surface associated antigens would minimize the non-specific and intolerable effects of cytotoxic agents on normal cells. The binding of ADCs to specific tumor associated antigens can trigger internalization of the binding complex and release the drugs inside the cancer cells for the intended cytotoxic action. The main difference between ADCs and fusion proteins is in the molecular structure, In ADSc, toxic agents are chemically conjugated to antibodies or their fragments while for fusion proteins, effector molecules are inserted into the protein on the genetic level. Goswami (Antibodies 2031, 2, 452-500)

Monoclonal antibodies have been demonstrated to be a promising carrier to deliver drugs, such as anti-tumor agents, to specific tissues (e.g., tumor tissues). Such compounds are typically referred to as toxin, radionuclide, immunoconjugates, immunotoxins, radioimmunoconjugates, and drug conjugates. Tumor cell killing occurs upon binding of the drug conjugate to a tumor cell and release and/or activation of the cytotoxic activity of the drug.

Challenges/Pitfalls

Although such efforts have been made for many years, problems which can occur are toxicity of the conjugated drugs to normal tissues as well as poor specificity and affinity of the mAb. Furthermore, even though conjugate is prepared to maintain its specificity and affinity, cytotoxic potency may be affected because covalently linked drugs are structurally altered by the coupling chemistry, and their original state may not be restored even after cleavage form the antibody. Therefore, one of the most important points for the preparation of desired immunoconjugates is control of the release of the agents at desired tumor sites. One approach is to use a linker which can be cleaved to release the toxin when the conjugate is internalized into the acidic compartment in tumor cells. The peptidyl linker, which can be recognized by tumor specific proteaseses, is cleaved, releasing the anti-tumor agent.

Considerations in Design of Antibody Drug Conjugates (ADCs)

Development of antibody-durg conjugates with therapeutic potential involves the optimization of several critical parameters. These include the use of highly potent drugs that are attenuated and stable while attached to the mAbs and the use of drug-mAb linkers that allow for the release of active drug only when the mAb has reached the target site. Another key factor is the choice of target antigen. The target antigen should internalize upon mAb binding, have high expression on tumor cells, and little to no expression on normal cells. Hambelt (Clinical Cancer Research, 10, 7063-7070, 2004).

Drug Loading (Drug to Antibody Ratio or DAR): is an important design parameter of antibody conjugates. An antibody-drug conjugate consisting of monomethyl auristatin E (MMAE) conjugated to the anti-CD30 mAb cAC10, with eight drug moieties per mAb was shown to have potent cytotoxic activity against CD30_ malignant cells. However, by reducing the quantity of MMAE from eight to four molecules per mAb, equivalent antitumor activity in vivo was obtained. Reducing to two MMAE molecules per antibdoy further reduced the in vitro activity but E2 had equivalent or better efficacy than E4 and E8 at doulbe the dose. Hambelt (Clinical Cancer Research, 10, 7063-7070, 2004)

One of the most important quality attributes of an ADC is the average number of drugs that are conjugated because this determines the amount of “payload” that can be delivered to the tumor cell and can direclty affect both safety and efficacy. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Examples:

Antibody-Drug Conjugates (ADCs) (immunoconjugates): are becoming an increasingly important class of therapeutic agents for treatment of cancer. ADC products in late stage clinical development include brenruximab vedotin (SGN-35; Seattle Genetics) for treatment of CD30 positive malignancies such as Hodgkin’s lymphoma, inotuzumab ozogamicin (CMC-544; Pfizer) for CD22 positive B cell malignancies such as non-Hodgkin lymphoma and trastuzumab emtansine (T-DM1; Genentech/Roche/ImmunoGen) for human epidermal growth factor receptor 2 (HeR2) positive metastatic breat cancer. ADCs harness the selectivity of mAbs to achieve targeted delivery of cytotoxic drugs. ADCs include the mAb which is specific to a tumor antigen, a cytotoxic agent and a linker species that enables covalent attachment of the cytotoxin to the mAb through either the protein or the glycan. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Examples of cytotoxic drugs that have been conjugated to mAbs include those that bind DNA (e.g., doxorubicin), alkylate DNA (e.g., calicheamicin, duocarmycin) or inhibit tubulin polymerization (e.g., maytansinoids, auristatins). The ADCs farthest along in clinical development contain boudn maytansines, auristatins and calicheamicins. (Jacobson, “Analytical methods for physicochemical characterization of antibody drug conjugates” mAbs 3:2, 161-172, 2011).

Antibody-Complement inhibitors: 

(He (ProQuest Dissertations and theses, 2004) describe using a single chain antibody variable fragment as a targeting moeity for the complement regulators Crry and CD59 to the rat kidney. The specific scFv was specific for rat kidney glomerulus and proximal tubulus.

Taube (“inhibition of complement activation decreases airway inflammation and hyperresponsiveness” Am. J. Resp. and Critical Care Med., (2003)) teach a recombinant soluble form of the mouse membrane complement inhbitor complement receptor-related gene y (Crry) fused to the IgG12 hinge CH2 and CH3 domains (Crry-Ig) significantly prevented the development of airway hyperresponsivenss, decreased airway and lung eosinophilia as well as the numbers of lung lymphocytes, decreased levels of IL-4, IL-5 and IL-13 in bronchoalveolar lavage fluid and decreased serum ovalbumin specific IgE and IgG1 suggesting that prevention of complement activaiton may have a therapeutic role in the treatment of allergic airway inflammation and asthma in sensitized individuals.

–Fusion anti-C3d + Complement Inhibitor

Holers (US 20200181249) discuses a fusion protein that includes anti-human complement c3d antiboy and a complement modulator polypeptide for treatment of complement-associated diseases.

–Fusion anti-annexin antibody + Complement Inhibitor:

Thurman and Holers  (14/571,102, published as US 10233235; see also US 16/015,019, published as US 2019/0002541) disclose fusion constructs of an anti-annexn A2 antibody and a complement inhibitor such as DAF, MCP, CD59, Crry and CR1.

Thurman (WO 2014/116880) disclsoes a targeted delivery that uses an antibody that recognizes an eptiope found to be present at sites of inflammation such as Annexin IV or phospholipid.

Antibody-Tumor toxic agent conjugates/Antibody Drug Conjugates (ADCs):  See also Conjugation and Liners and Clevable linkers in outline.

The Toxin:

Of 47 ADC candidates in clinical studies as of 2014, 16 candidates used maytansinoids, while 22 candidates use auristatins. In most cases, a license or a purchase of the respective toxin, from ImmunoGen or Seattle Genetics, respectively, would be necesary, because tehse 2 companies are the major IP holders regarding these toxins or toxin-linker combinations. (Storz, “Antibody-drug conjugates: Intellectual property considerations” mAbs 7:6, 989-1009, 2015)

The cytotoxins used in ADCs have been used as solo chemotherapeutic agents themselves. The selected drug should be toxic at a subnanomolar concentraiton becasue the effiiceincy of delivering it to target cells is low. To date, two general classes cf cytotoxins ahve been used in ADC design; microtubule inhibitors such as auristatins (e.g., monomethyl auristatins E and F) and maytansinoids and DNA damaging agents such as calicheamicins, duacarmysins, doxorubicin and pyrrolobenzodiazepines. A third class of cytotoxins, topoisomerase 1 inhibitors is the most recent to be approved by FDA. (Chamow “Manufacturing challenges of therapeutic antibody drug conjugates” BioProcess international 21(9) 2023). 

The difference between an efficacious dose adn one that causes damage to normal tissues defines a drug’s therapeutic window. A drug candidate is conisdered to be promising if that difference is large. Traditional chemotherrapies have narrow windows because they are inherently cytotoxic and lack specificity for tumor cells. By contrast, incorporating those same chemotherapeutic agents as ADC payloads can devliver them to tumors selectively, broadening their therapeutic index. The reuslting ADCs can be efficacious at lower doses and across broader dose ranges than those of the same cytotoxins adminstiered alone as chemotherapeutics. Chamow “Manufacturing challenges of therapeutic antibody drug conjugates” BioProcess international 21(9) 2023).

—Maytansinoids: If administered in unconjugated form, maytansinoids can cause adverse efects to the central nervous sytem and gastrointestinal tract. In conjugate form, maytansinoid is inactive and can be administered without causing systemic toxicity to a patient. After binding to the surface of a tumor cell, the conjugate is internalized and the maytansinoid is released from the antibody and can exert its cytotxic effect on the tumor cells. The maytansinoids are anti-mitotic drugs 100-1000 fold more cytotoxic than conventional cancer chemotherapeutic agents such as methotrexate, daunorubicin and vincristine.

Preparation of maytansanoid derivatives with cleavable but highly stable linkers to monoclonal antibodies has been disclosed in US 5,208,020. The synthesis of disulfide and thiol containing maytansinoids which can be linked to cell binding agents via disulfide or any other sulfur containing link such as theioether or thioester links is disclosed in US 5416064.

Cari (US 5,208,020) discloses a cytotoxic agent comprising one or mroe maytansinoids linked to a cell binding agent.

Antibody-Nanoparticle-Active Agent conjugates:  Lanza (US2003/0086867) teaches that nanoparticles can be made target-specific to blood clots or thombosis, especially in vivo, by directly coupling the nanoparticles to a targeting ligand such as an antifibrin antibody.

Antibody-immunoadhesin chimera: comprises a molecule which combines at least one binding domain of an antibody with at least one immunoadhesin. For more on immunoadhesins see left

Diptheria toxin: Chemotherpaeutic agents useful in the generation of such conjugates include enzymtically active toxins such a diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, maytansinoids (mitototi inhibitors which act by inhibiting tubulin polymerization). See (Nadarajah, US14/355818 (US2014/0301977)) for list of chemotherpaeutic agents. hand panel.

Probody Therapeutics (Activatable Antibodies): (see cleavable linkers)