Year of the ox

High levels of human polyclonal antibodies have been produced in a transgenic large animal.

With a market now worth well over $2 billion in the United States¹, human polyclonal antibodies purified from thousands of plasma pools have become standard therapy for many viral infections and immune disorders² and for neutralization of toxins³. Despite their clinical potential, however, the use of polyclonal antibodies remains limited by issues related to their supply, cost and safety⁴. In this issue, Kuroiwa et al.⁵ bring us a step closer to large-scale production of relatively homogenous recombinant polyclonal antibodies, which could alleviate these problems and expand application of this therapeutic modality to new indications⁶.

Unlike monoclonal antibodies, which recognize a single epitope, polyclonal antibody preparations bind multiple epitopes on the disease-causing agent and can thereby neutralize distinct variants of toxins or infectious particles, making them the agents of choice for treating certain medical emergencies and acute illnesses. Hyperimmune globulins—sourced from human or animal donors with high titers of antibodies against specific antigens—are in high demand to curb immunosuppression associated with transplants; prevent Rh hemolytic disease; treat and prevent infections such as hepatitis B, hepatitis A, rabies, respiratory syncytial virus, cytomegalovirus and varicella-zoster; and neutralize toxins, including diphtheria, botulism, digoxin and snake and spider toxins^2,3.

The ability to produce human antibodies in mice expressing human immunoglobulin genes has long been appreciated⁷, and mice now provide a convenient source of hybridomas for generating candidate therapeutic human monoclonal antibodies. However, the small body size of mice makes them unsuitable for synthesizing large amounts of hyperimmune globulins. Aiming to translate this approach to a large animal, Kuroiwa and colleagues previously expressed the human immunoglobulin heavy chain and λ-light chain from a human artificial chromosome in cloned cows⁸. Although these animals did produce human antibodies, the levels were too low to be of practical utility as the active endogenous immunoglobulin loci suppressed expression of the human genes^6,8.

The feasibility of introducing human immunoglobulin genes and knocking out endogenous immunoglobulin genes in cattle has been far from certain. First, it is not straightforward to perform multiple genetic modifications in large animals: embryonic stem cell lines are not available and generation intervals are long (around three years in cattle). Successive rounds of transfection and selection in primary cells, which have a limited life span, each followed by somatic cell nuclear transfer (to regenerate the cell line) are necessary to introduce the targeting constructs and the human immunoglobulin loci. Second, the accumulation of epigenetic errors caused by successive rounds of nuclear transfer has been reported to compromise the viability of offspring. Finally, it has been unclear whether human immunoglobulins alone could support bovine humoral immunity in the absence of endogenous bovine immunoglobulins.

These formidable challenges make the achievement of a transgenic calf expressing high levels of human antibodies all the more remarkable. Kuroiwa et al.⁵ focused first on successively inactivating all the bovine IgM heavy chain genes (Fig. 1), whose expression is essential for B-cell development. Because ruminants, unlike mice and humans, have two functional IgM loci (IGHM and IGHML1), four alleles had to be targeted to knock out IgM heavy-chain production. In the next step, the IGHM^−/− IGHML1^−/− fibroblasts were transfected with an artificial human chromosome (κHAC) bearing the unrearranged human heavy and κ-light-chain loci (Fig. 1). In the end, after seven rounds of cloning, a healthy transgenic calf, with all bovine IgM heavy chain alleles inactivated and bearing the human artificial chromosome, was obtained. This animal expressed 60-fold more human immunoglobulins than animals described previously⁸—a yield that is potentially competitive from a cost perspective with producing nonhuman hyperimmune globulins.

Figure 1: Cattle capable of producing human polyclonal antibodies are produced by multiple cycles of transfection, selection and nuclear transfer⁵.

Kim Caesar

Each of the four bovine IgM heavy chain alleles is knocked out by homologous recombination followed by somatic cell nuclear transfer to extend the life span of the selected cell lines. An artificial chromosome carrying the human immunoglobulin heavy and κ-light chain loci (κHAC) is transferred to the multitargeted bovine cell lines by microcell-mediated chromosome transfer, and three additional nuclear transfer steps are performed to obtain a healthy transgenic calf with the κHAC/IGHM^−/−/IGHML1^−/− genotype. Vaccination of this animal with an antigen of interest produces ∼20% fully human antibodies and ∼80% chimeric antibodies (bearing human heavy chains and bovine light chains).

Vaccination of the calf with anthrax-protective antigen yielded high titers of anthrax-specific immunoglobulins. Although ∼80% of serum IgGs were functional chimeric antibodies comprising human heavy chains and bovine light chains, the remainder were fully human (Fig. 1). Once purified, the hyperimmune globulins fully protected mice challenged with anthrax spores and in an in vitro toxin neutralization assay outperformed a control anthrax hyperimmune globulin preparation derived from human donors.

Importantly, the calf's immunization response was similar to that of wild-type cattle, confirming that the human immunoglobulin loci can support the humoral response in the absence of bovine IgM. Other transgenic calves produced in this study appeared to produce similar levels of human IgGs, suggesting that a herd of cattle with this genotype could provide an abundant source of human hyperimmune globulins. Nevertheless, extensive purification will be necessary to obtain preparations containing only fully human IgGs, which will increase production costs. Knocking out the bovine Igλ locus, which contributes ∼90% of light chains in cattle, in this line could further increase the proportion of fully human immunoglobulins and improve process yields.

Although the seven years that have elapsed since this group reported transchromosomic calves expressing human immunoglobulin loci⁸ might seem like a long development time, it is worth remembering that the line generated in the present study⁵ or subsequent lines could support production of multiple products. Each new hyperimmune globulin product would be dependent on the antigen used in the immunization protocol, rather than the bovine line or the purification process. Scale-up should be relatively straightforward, although this might require cloning rather than natural breeding.

It is still too early to confidently predict the commercial success of human hyperimmune globulins from transgenic cattle. Uncertainties remain concerning, for example, the impact of purification on production costs and the feasibility of using somatic cell nuclear transfer to generate large numbers of animals. Clinical studies—the costliest and riskiest aspect of drug development—must also be completed. But given the flexibility and scalability of using transgenic large animals, this approach may be well placed to compete with traditional human- and animal-derived intravenous immunoglobulins, hyperimmune globulins, and monoclonal and polyclonal antibodies produced in cell culture⁹ in applications spanning infectious diseases, oncology, neurological conditions and immune modulation. As we enter the Chinese year of the ox, it seems fitting to look forward to clinical trials of polyclonal antibodies obtained from transgenic cattle.