PEG & ADC

ADC to XDC, Five Major Elements of Antibody Conjugate Drugs, Exploring the Fundamental Source of Innovation

From ADC to XDC, the future trend of conjugated drugs will focus on new target antigens, payloads with new mechanisms of action, new antibodies and carrier forms, etc. Target indication selection + optimal structural combination are the current focus of research and development.

Antibody-Drug Conjugate (ADC) is a type of conjugated tumor-targeted antibody (Tumor Targeted Antibody) and small molecule cytotoxic drug (Cytotoxic Drug) using specific coupling technology through a linker. drug.

Many articles have introduced antibody conjugated drugs from various dimensions. This article will once again outline the five basic elements of antibody conjugated drugs (antigen target, antibody, linker, payload and conjugation method).

Mechanism:

ADC drugs have antibodies that recognize tumor antigens with high specificity. After intravenous injection, the drugs pass through the blood circulation, distribute to tumor tissues and bind to tumor surface antigens.

The complex of ADC and antigen internalizes the payload carried by it into tumor cells through endocytosis, and is transported to lysosomes and released in a highly active form. It induces cancer cell apoptosis through DNA damage or inhibition of microtubule synthesis.

1. Target

The starting point for ADC preparation is target selection. Drug safety and effectiveness mainly depend on the selection of target antigen and its interaction.

Factors to consider:

(1). Whether the target antigen is highly expressed in tumors but has no or low expression in normal cells;

(2). Whether the target antigen is distributed on the cell membrane surface of tumor cells so that it can be recognized by specific antibodies;

(3). Whether the antigen is not easy to fall off to prevent the antibody from binding to the antigen in the circulation;

(4). Whether the target antigen has internalization properties.

More than fifty antigens have been used as target antigens recognized by ADCs. For the selection of target antigen sites, scientists are exploring different design strategies.

Important target antigens in tumor cells/tumor microenvironment

1. Target antigens overexpressed in cancer cells (distribution of cancer cells):

GPNMB,CD70,CD56,Trop-2,FRα,Tissue factor,ENPP3,p-cadherin,mesothelin,STEAP1,CEACAM5,Mucin 1,Nectin 4,SLC44A4,PSMA,LIV1,5T4,SC-16,Guanylyl cyclase C;

2. Driving oncogenes (distribution of tumor-associated fibroblasts) HER2,EGFR

3. Target antigen (distributed proteoglycan) in tumor vasculature

EDB,ETB,PSMA VEGFR2,ROBO4,Tissue factor

4. Target antigen in the matrix (distribution of collagen)

Tenascin C、Collagen IV, Periostin

5. Target antigens in hematological malignancies (distributed in blood cells)

CD22,CD30,CD33,CD79b,CD19,CD138,CD74,CD37,CD19,CD98

CD22,CD30,CD33,CD79b,CD19,CD138,CD74,CD37,CD19,CD98

At present, the ADC target, HER2, is becoming more and more intense. Three HER2 ADCs have been approved for marketing around the world, and the proportion of HER2 that has entered the clinical stage globally accounts for more than 20%.

Targets such as TROP-2, EGFR, and Claudin18.2 are also under research by many companies, and Chinese companies have entered the forefront of global research and development. In addition, there are more than 400 preclinical ADC drugs in the world, and the preclinical target layout is relatively scattered.

TROP2, one ADC (gosatuzumab) has been approved in the world, and there are many domestic pipelines under development.

Nectin-4, one ADC (veentuzumab) has been approved for marketing in the world.

There are more than ten CLDN18.2 ADCs entering the clinical stage around the world, most of which are in early clinical stages.

The ADC targets presented at the 2023 ESMO Conference are diverse. In addition to traditional targets, new targets such as PTK7 and FolRα have also begun to emerge.

2. Antibody

Antibodies are an important part of ADC design. They should be able to specifically recognize the target antigen of tumor cells and have high affinity with the target antigen.

Antibodies should also have the characteristics of weak immunogenicity, long half-life, promoting effective internalization, and good blood circulation stability.

Antibodies can be divided into five categories according to their heavy chain constant region sequences: IgG, IgA, IgD, IgE and IgM.

Currently approved ADCs basically use immunoglobulin G (IgG) antibodies, with subtypes including IgG1, IgG2, IgG3 and IgG4.

Among them, IgG1 is the most used subtype in conjugation research and development due to its moderate molecular weight, long half-life, high affinity, easy preparation, and strong Fc effector function.

IgG1 has the highest content in serum and can induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), etc. through high binding affinity to Fc receptors. Strong effect function.

The antibody portion of the ADC can take many forms, such as bispecific antibodies or single domain antibodies. BisAbs can target two different epitopes of the same target antigen, or they can target two different target antigens.

ADCs can also use single-domain antibodies coupled with killer radioactive elements or toxic molecules to develop new conjugated drugs.

3. Linker

The linker is an important component of the ADC and connects the antibody to the payload.

The stability of the linker in the blood is very important. When the ADC enters tumor cells or is transported to lysosomes, the linker should rapidly break down to release the payload.

Linkers can affect drug-to-antibody ratio (DAR), payload release time, therapeutic index, and pharmacokinetics/pharmacodynamics.

Connectors can be divided into two types: cleavable and non-cleavable.

Common linkers include: valine-citrulline (VC) linker, N-succinimide 4-(2-pyridyl disulfide) butyrate (SPDB) linker, hydrazone linker, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide (SMCC) linker, maleimidocaproyl (MC) linker, N-succinimidyl- 4-(2-pyridyl disulfide) valerate (SPP) linker, thioether linker, tetrapeptide linker and carbonate linker, etc.

1. Cuttable connectors

Cleavable linkers are cleaved by a variety of mechanisms, including acid-labile cleavage of hydrazone bonds, reductive cleavage of disulfide bonds, and enzymatic cleavage of peptide bonds.

Cleavable linkers do not necessarily produce bystander effects and depend mainly on the membrane penetration and charge properties of the released payload.

2. The connector cannot be cut

ADCs with non-cleavable linkers will only release their payload after entering the lysosomes of tumor cells and being degraded by proteases.

After the non-cleavable linker is endocytosed into the lysosome, the linker will not be degraded, and the connected antibody will be degraded into amino acids, forming an amino acid-linker-small molecule cytotoxic complex.

This type usually does not produce bystander effects because the “linker-amino acid residue” is charged, which limits its membrane penetration and diffusion.

Linker optimization is mainly to increase stability in plasma and adapt to the bystander effect; as well as to adapt to toxins with different mechanisms of action. Cleavable linkers have become the mainstream trend in current ADC applications. However, due to the strict requirements of high toxic loads on cycle stability, “enzyme-sensitive peptide technology” has gradually replaced “acid-sensitive linker technology”.

4. Payload

ADC payload is its most important effect component. Commonly used molecules include: microtubule formation inhibitors, DNA damage factors, and DNA transcription inhibitors.

Microtubule formation inhibitors prevent the polymerization of microtubules by binding to microtubules, thereby arresting the cell cycle, producing cytotoxicity, and exerting anti-tumor effects.

Such as: au⁃ ristatin, maytansine and its analogs.

DNA damage factors bind to the minor groove of DNA and promote alkylation, breakage, or cross-linking of DNA strands.

Such as:Calicheamicin、Duocarmycins、 Anthracyclines、Pyrrolobenzodiazepine dimers.

DNA transcription inhibitors

Such as: Quinoline Alkaloid (SN-38) andAmatoxin, etc.

Other new types: apoptosis inducers, RNA splicing inhibitors, nicotinamide phosphoribosyltransferase inhibitors, etc.

As technology development continues to advance, more and more payload types are being explored.

It is difficult to restructure existing toxins to improve efficacy. The main purpose is to expand ADC treatment options and treatment windows through the diversification of toxins.

Anthracyclines based on topoisomerase 2 inhibitors and α-Amanitin based on RNApol II inhibitors have good preclinical data.

5. Conjugation

Coupling methods include non-directed coupling and fixed-point coupling.

Non-site-specific coupling methods mainly include lysine and cysteine coupling.

Site-specific coupling uses genetically engineered sites for specific coupling to achieve a more uniform ADC, which can connect cytotoxins at specific sites.

1. Non-fixed point coupling

The coupling selectivity is poor and the product uniformity is poor.

The earliest use of electrophilic groups, such as maleimide or N-Hydroxy Succinimide (NHS), to react with the amino group of exposed lysine. It shows randomness, the number of drug molecules carried on the antibody is different, and the product heterogeneity is large, affecting parameters such as PD/PK.

Non-site-directed coupling of cysteine ​​that reduces disulfide bonds is a commonly used coupling method. Enhertu, Trodelvy, and Adcetris all use this method.

Monoclonal antibody IgG1 contains 12 intrachain disulfide bonds and 4 interchain disulfide bonds.

Intrachain disulfide bonds are located between the two antiparallel β-sheet structures of the antibody, resulting in lower reactivity.

The interchain disulfide bond is easily acted upon by reducing agents, which can reduce 8 sulfhydryl groups and further couple with the toxin molecules connected to the reactive groups, forming 0, 2, 4, 6, and 8 major drug coupling forms.

2. Fixed point coupling

Fixed-site coupling is the future research and development trend of ADC. Fixed-site coupling technology can obtain ADC drugs with better uniformity, improve the stability of ADC drugs, and reduce off-target toxicity.

Site-directed coupling technology mainly includes: specific site coupling technology, non-natural amino acid coupling technology, glycan coupling technology and short peptide tag coupling technology, etc.

Specific site coupling technology, such as Genentech’s Thiomab technology, inserts cysteine residues at specific positions of the antibody through genetic engineering technology, coupling the sulfhydryl group on the cysteine with small molecule poisons, forming a stable DAR value ADC drugs with high uniformity.

Compared with ADC drugs obtained by traditional random coupling, ADC drugs based on this technology have low aggregation, high stability in plasma, and strong anti-tumor activity in vivo and in vitro.

Technology based on non-natural Amino Acid (nnAA) provides site-specific solutions for the preparation of ADC.

Compared with natural amino acids, nnAA is artificially synthesized with better specificity and stability. nnAA is introduced at specific sites of proteins through orthogonal tRNA synthetase, orthogonal tRNA, and a unique codon system.

GlycoConnect technology utilizes the glycosylation modification of asparagine 297 residue in the Fc segment of most IgG monoclonal antibodies, which is a very suitable specific drug conjugation site.

The antibody excises the N-terminal glycan by endoglycosidase, leaving glycosamine (GlcNAc), and adds N-azidoacetylga-lactosamine (GalNAz).

Short peptide tag conjugation technology couples cytotoxins to specific short peptide tags containing 4 to 6 amino acid residues.

Introducing unique short peptide tags into antibodies for enzymatic modification in vivo or in vitro. Allows specific amino acids in the peptide tag to be functionalized and coupled with drug linkers.

The glutamine tag (Leu-Leu-Gln-Gly, LLQG) is introduced into the antibody molecule, so that the glutamine in the tag is recognized by mTG, and the amine-containing drugs are transferred.

At the N-terminus or C-terminus of the antibody, a short peptide tag LCx⁃ PxR recognized by Formylglycine-Generating Enzyme (FGE) is inserted.

After the antibody is co-expressed with FGE, the cysteine ​​in the short peptide tag is oxidized by intracellular enzymes to aldehyde-containing formylglycine, which can be coupled with amino functionalization reagents.

The introduction of short peptide tags located in different regions of the antibody may lead to immunogenicity.

From ADC to XDC

ADC drug technology, through the design of original drugs, has been expanded to XDC all-things coupling. At present, peptide-drug conjugates and antibody-radionuclide conjugates have attracted much attention, and a variety of other new conjugated drugs are also being developed at an accelerated pace.

ADC drugs create technology by disassembling and optimizing five core elements, screening for highly expressed target antigens, antibodies with balanced specificity, metabolically stable linkers, payloads with high anti-tumor activity, and conjugation technology with uniform DAR values. barrier.

With the rapid advancement of multiple new conjugate drugs, targeted drugs will also enter a new era.